Recombinant virus vectors encoding human papillomavirus proteins

ABSTRACT

The invention provides a recombinant virus vector for use as an immunotherapeutic or vaccine. The recombinant virus vector comprises at least one pair of nucleotide sequences heterologous to the virus and which have sufficient sequence homology that recombination between them might be expected. The pair of nucleotide sequences are arranged in the virus vector such that they are inverted with respect to each other. The virus vector is able to infect a mammalian host cell and express as polypeptide the heterologous nucleotide sequences in the host cell. For infection thought to be caused by HPV infection, the pair of nucleotide sequences encode part or all of human papillomavirus (HPV) wild-type proteins or mutant proteins immunologically cross-reactive therewith. For an immunotherapeutic or vaccine against cervical cancer, the recombinant virus vector encodes part or all of the HPV wild-type proteins HPV16E7 and HPV18E7 or mutant proteins immunologically cross-reactive therewith.

FIELD OF THE INVENTION

This invention relates to recombinant virus vectors. In particular, itrelates to recombinant virus vectors designed to overcome the problem ofrecombination between homologous nucleotide sequences. It also relatesto recombinant virus vectors encoding human papillomavirus proteins; toimmunotherapeutics and vaccines for conditions associated with HPVinfection; to the production of a virus (e.g. vaccinia virus) engineeredto express antigens encoded by human papillomavirus types 16 and 18 andto immunotherapeutics and vaccines for cervical cancer.

BACKGROUND OF THE INVENTION

In recent years, strong evidence has been adduced for a link betweencervical carcinoma and infection with certain types of humanpapillomavirus (HPV), particularly with types 16, 18, 31, 33 and 35(Gissman et al., Cancer Cells 5,275, 1987). This is based onhybridisation studies which have indicated that more than 85-90% ofbiopsies from cervical tumours can be shown to contain papillomavirusDNA. HPV16 DNA is most commonly found (in about 60% of tumours) withHPV18 the next most frequent (about 20%) and the other types accountingfor a further 5-10%. In many instances, tumour cells from the biopsiesdo not however, contain the complete genome, but rather a deleted form.The extent and location of the deleted information within the virusgenome is variable, but a general feature is the retention of the partof the genome encoding the E7 protein (Schwarz et al., Nature 314, 111,1985). In addition, the adjacent E6-encoding region is usually present.The ubiquitous presence of the E7-encoding region in tumour cellssuggests that the protein product of this gene might play a role in theinduction or maintenance of the transformed phenotype. Indeed in mostcell lines established from tumour biopsies, expression of the E7 genecan be detected (Smotkin & Wettstein, PNAS, 83, 4680, 1986).Furthermore, it has been shown that the E7 gene product can bind to theretinoblastoma (Rb) gene product, a recognised "anti-oncogene" in normalhuman cells (Munger et al., EMBO J. 8,4099, 1989). This strengthens thebelief that E7 is directly involved in cell transformation.

The presence and expression of the E7 and E6 genes in tumour cellsderived from cervical carcinoma biopsies, suggests the possibility thatthese proteins could be potential targets for the immunologicalrecognition of the tumour cells. It is well known that viral proteinsproduced inside mammalian cells can be processed through a host cellpathway to short peptides, which then form a complex with host MajorHistocompatibility Complex (MHC) Class 1 molecules and are transportedto the cell surface. These complexes may then present a target forrecognition by the host immune system. Interaction of the complex withthe receptor molecule on the surface of cytotoxic T cells (the T cellreceptor) can then lead to activation of the T cells to proliferate orto destroy the recognised cell. It is possible, therefore, that thepresence in the body of a population of cytotoxic T lymphocytes (CTLs)which are capable of recognising cells expressing the HPV E6 and/or E7proteins could afford protection against the development andproliferation of cervical tumours. Indeed it has been reported thatnormally oncogenic mouse cells engineered to express the HPV E7 proteinare unable to form tumours in mice which have been previously immunisedwith non-tumorigenic E7-expressing cells, and that this rejection ismediated by CD8+lymphocytes (CTLs) (Chen et al., PNAS 88, 110, 1991).Further, the generation of an active population of such cells subsequentto tumour initiation could result in regression of the tumour.

There are numerous reports on the construction of recombinant virusese.g. vaccinia viruses containing, and expressing foreign genes (Mackett& Smith, J. gen. Virol. 67,2067, 1986), and several reports of the useof these recombinant viruses to generate effective immune responsesagainst the expressed foreign antigens. A particular advantage of thisroute for delivery of antigens for vaccination is that it may lead tothe development of cellular as well as humoral immunity. This is becausethe foreign proteins will be produced inside cells of the infectedindividual in a manner similar to that which occurs during naturalinfection. This means that they should be processed through the correctpathway to allow generation of a CTL response. In several cases, it hasbeen demonstrated directly that immunisation with the recombinant virusis capable of producing a cellular immune response in the form offoreign antigen-specific CTLs (Moss & Flexner, Ann. Rev. Immunol.,5,305, 1987). Furthermore, vaccination of animals with recombinantvaccinia viruses expressing certain tumour-specific antigens, such asthe human melanoma-associated antigen P97 (Estin et al., PNAS, 85, 1052,1988), the bovine papillomavirus E7 protein (Meneguzzi et al., Vaccine,8, 199, 1990) and the human breast cancer-associated antigen ETA(Hareuveni et al., PNAS, 87, 9498, 1990) has been demonstrated to resultin the induction of immunity against tumour initiation and progression.

SUMMARY OF THE INVENTION

The present applicants have recognised the desirability of producing arecombinant virus vector which is useful as an immunotherapeutic orvaccine for conditions caused by HPV infection, for example for cervicalcancer. With respect to cervical cancer, the art at the time of theapplicants making the present invention recognised the E7 gene as havingthe potential to immortalise cells. Therefore, it would be feltinappropriate to incorporate the E7 gene in an immunotherapeutic. Theapplicants however, have recognized the surprising usefulness ofincluding the E7 gene in an immunotherapeutic. They have also recognizedthat the beneficial effects to be gained by treatment with animmunotherapeutic comprising the E7 gene are likely to outweigh by farany risk associated with the oncogenic activity of the E7 gene. Thus, anaspect of the applicants invention involves the use of a recombinantvirus vector which expresses an E7 gene, as an immunotherapeutic orvaccine. Furthermore, the applicants provide embodiments of theirinvention in which these risks are reduced still further by specificalteration of the gene sequences in order to reduce the oncogenicpotential of the E7 gene without compromising its ability to stimulatean appropriate immune response.

The present applicants have also recognized that where a number of HPVproteins which may be encoded by different HPV strains are implicated asbeing associated with a particular HPV-associated condition (forexample, cervical carcinoma, HPV16 and HPV18; genital warts, condylomaacuminata, respiratory papillomatosis, HPV6 and HPV11; squamous cellcarcinoma in immunosuppressed individuals, HPV5 and HPV8), rather thanproduce a plurality of recombinant viruses engineered separately toexpress each of the implicated proteins, it would be advantageous toproduce a single virus recombinant which is able to express part or allof the sequences of more than one of the proteins. Thus, with respect tocervical cancer, rather than produce four recombinant viruses engineeredseparately to express each of the potential targets for immunologicalrecognition of cervical tumour cells i.e. the HPV16 E6, HPV16 E7, HPV18E6 and HPV18 E7 proteins, it would be especially advantageous to producea single virus recombinant which is able to express the part or all ofthe sequences of more than one of the proteins, preferably at least twoof the proteins and most preferably all four proteins. That the presentapplicants are able to achieve this is particularly surprising. This isbecause, the coding sequences for many HPV proteins are highlyhomologous to other equivalent HPV proteins (for example from othervirus strains). Thus, the HPV16 E6 and HPV18 E6 proteins show overallhomology of 62% and comprise regions of very high homology. The same istrue for HPV16 E7 and HPV18 E7 which show overall homology of 57%, withparticular regions of very high homology. This means that one wouldexpect recombination to create problems such as loss of gene sequences.The applicants have, however, devised a novel strategy designed tominimise the likelihood of such recombination events and to circumventthe deleterious effect of those events should they indeed arise. Thus,surprisingly, the invention provides recombinant virus vectors whichcomprise at least one pair of nucleotide sequences which have sufficientsequence homology that recombination between them might be expected. Theat least one pair of nucleotide sequences may encode part or all ofhuman papillomavirus (HPV) wild-type proteins or mutant proteinsimmunologically cross-reactive therewith. In particular, the inventionprovides a recombinant vector which can maintain stably, and express,part or all of four of the desired gene sequences from HPV16 and HPV18.

Thus, the present invention provides a recombinant virus vector for useas an immunotherapeutic or vaccine which comprises at least one pair ofnucleotide sequences heterologous to said virus and which havesufficient sequence homology that recombination between them might beexpected wherein said pair of nucleotide sequences are arranged in saidvirus vector such that they are inverted with respect to each other toreduce the likelihood of recombination events leading to loss of part orall of said sequence and said virus vector is able to infect a mammalianhost cell and express as polypeptide the heterologous nucleotidesequences in said host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the present invention is more fully understood anembodiment will now be described in more detail with reference to thefigures in which:

FIG. 1 (Parts a and b): FIG. 1(a) (SEQ ID NOS:7-10) shows the nucleotidesequence and three-frame translation of HPV16 E6/E7 polymerase chainreaction product (underlined regions indicate the E6 and E7 codingsequences); FIG. 1(b) (SEQ ID NO:11-14) shows the nucleotide sequenceand three-frame translation of HPV18 E6/E7 polymerase chain reactionproduct (underlined regions indicate the E6 and E7 coding sequences);

FIG. 2 shows the cloning and modification of the HPV16 and HPV18 E6 andE7 Genes;

FIG. 3 shows an open reading frame plot of vaccinia virus from positions17201-18450 of the region covered by the four fragments SalF,G,H and I;short vertical lines denote termination codons, lines topped with boxesdenote initiation codons, rectangles show relevant open reading frames,and arrows show direction of upper and lower DNA strands;

FIG. 4 shows an open reading frame plot of vaccinia virus from positions21001-22000 of the region covered by the four fragments SalF,G,H and I;short vertical lines denote termination codons, lines topped with boxesdenote initiation codons, rectangles show relevant open reading framesand arrows show direction of upper and lower DNA strands;

FIG. 5 shows an open reading frame plot of vaccinia virus from positions23501-25000 of the region covered by the four fragments SalF,G,H and I;short vertical lines denote termination codons, lines topped with boxesdenote initiations codons, rectangles show relevant open reading framesand arrows show the direction of upper and lower strands of DNA;

FIG. 6 shows a codon usage plot of vaccinia virus from positions17201-18450 of the region covered by the four fragments SalF,G,H and I;arrows show direction of each DNA strand;

FIG. 7 shows a codon usage plot of vaccinia virus from positions21001-22000 of the region covered by the four fragments SalF,G,H and I;arrows show direction of each DNA strand;

FIG. 8 shows a codon usage plot of vaccinia virus from positions23501-25000 of the region covered by the four fragments SalF,G,H and I;arrows show direction of each DNA strand;

FIG. 9 (SEQ ID NOS:15-17) shows the DNA sequence around site A showingtranslations in single letter amino acid code of genes SalF 17R and SalF19R;

FIG. 10 (SEQ ID NOS:18-20) shows the DNA sequence around site B showingtranslations in single letter amino acid code of genes SalF 20R andSalF20.5R;

FIG. 11 (SEQ ID NOS:21-22) shows a comparison of the SalG2R open readingframe to the yeast guanylate kinase gene sequence;

FIG. 12 (SEQ ID NOS:23-25) shows the DNA sequence around site D showingtranslations in single letter amino acid code of genes HindB3R and HindB4R;

FIG. 13 shows the cloning of vaccinia virus (Wyeth strain) neutralsites;

FIG. 14 shows the cloning of vaccinia virus promoter sequences;

FIG. 15 shows the construction of vaccinia promoter-driven E6-7cassette;

FIG. 16 shows the cloning of the E6-7 cassette into vaccinia virus(Wyeth strain) neutral sites;

FIG. 17 is a diagram showing the recombination required to generate thefinal therapeutic vaccinia virus--HPV recombinant virus;

FIG. 18 (SEQ ID NOS:26-58) shows the synthetic oligonucleotides used inthe construction of the therapeutic vaccinia virus HPV recombinant;

FIGS. 19a and 19b show (SEQ ID NOS:59-62) the nucleotide sequence ofvaccinia virus (WR strain) from positions 17201-18450 of the regioncovered by the four fragments SalF,G,H and I;

FIGS. 20a and 20b show (SEQ ID NOS:63-66) the nucleotide sequence ofvaccinia virus (WR strain) from positions 21001-22000 of the regioncovered by the four fragments SalF,G,H and I;

FIGS. 21a, 21b and 21c show (SEQ ID NOS:67-70) the nucleotide sequenceof vaccinia virus (WR strain) from positions 23501-25000 of the regioncovered by the four fragments SalF,G,H and I.

FIG 22a shows the DNA fragments expected when the HPV DNA cassette isinserted at Site A (as marked on FIGS. 3 and 6) of recombiant vacciniavirus (V9a.1) and FIG. 22b depicts the PCR results;

FIG. 23 depicts Western blot analyis for the presence of HPV E67 fusionprotein expressed by recombinant virus vector;

FIG. 24a shows the PCR amplification reaction to confirm presence andarrangment of the inserted HPV sequences and FIG. 24b depicts the PCRresults;

FIG. 25 shows standard vaccina virus plaque assay results which showsthat the recombinant virus retains the ability to replicate mice; and

FIGS. 26a, 26b, 26c, 26d, 26e, 26f, and 26g show a variety of optionsfor arrangment of HPV16E6 and E7 and HPV18E6 and E7 coding sequences ina recombinant virus vector.

The at least one pair of nucleotide sequences may encode part or all ofhuman papillomavirus (HPV) wild-type proteins or mutant proteinsimmunologically cross-reactive therewith. The pair of nucleotidesequences may encode part or all of the protein E7 from both HPV16 andHPV18 or functional equivalents thereof. The pair of nucleotidesequences may encode part or all of the proteins E6 from both HPV16 andHPV18 or functional equivalents thereof.

The recombinant virus vector may comprise a further pair of nucleotidesequences heterologous to said virus and which (i) have sufficientsequence homology that recombination between them might be expectedwherein said further pair of nucleotide sequences are arranged in saidvirus vector such that they are inverted with respect to each other andsaid virus vector is able to infect a mammalian host cell and express aspolypeptide the further pair of heterologous nucleotide sequences insaid host cell.

The further pair of nucleotide sequences may encode part or all of HPVwild-type proteins or mutant proteins immunologically cross-reactivetherewith.

For example, the present invention also provides a recombinant virusvector which in addition to the E7 coding sequences, also comprises andis adapted to express genetic sequences encoding part or all of theprotein E6 from both HPV16 and HPV18 or functional equivalents thereof.The genetic sequences may comprise sequences encoding HPV16 E6/E7 andHPV18 E6/E7 as shown in FIGS. 1(a) (SEQ ID NOS:7-10) and 1(b) (SEQ IDNOS:11-14) respectively.

The genetic sequences may encode an antigenic moiety of the saidproteins.

Either or both of the nucleotide sequences in a pair of nucleotidesequences may be altered to make them less homologous than an equivalentpair of nucleotide sequences encoding wild-type HPV proteins. Thealteration in nucleotide sequence may be in an area of high sequencehomology Preferably, the alteration in nucleotide sequence will notresult in an alteration of the encoded amino acid sequence.

Two or more nucleotide sequences each encoding separate proteins may befused together to form a single open reading frame. Thus the geneticsequences encoding part or all of the proteins E6 and E7 from HPV16, maybe fused together to form a single open reading frame. The geneticsequences encoding part or all of the proteins E6 and E7 from HPV18, maybe fused together to form a single open reading frame. The geneticsequences encoding part or all of the proteins E6 and E7 from both HPV16and HPV18, may be fused together to form a single open reading frame.Thus, the recombinant virus vector may have the pairs of nucleotidesequences arranged according to any one of the options shown in FIG. 26.Where the recombinant virus vector comprises an open reading framehaving a fused genetic sequence encoding part or all of the proteins E6and E7 from HPV16, and a separate open reading frame having fusedgenetic sequences encoding part or all of the proteins E6 and E7 fromHPV18, the two open reading frames may be inverted with respect to oneanother. For example, the two open reading frames may be arranged in therecombinant virus vector adjacent to each other. The inversion may besuch that the E6 coding sequences of HPV16 and HPV18 are both locatedbetween the E7 coding sequences of HPV16 and HPV18. Alternatively, theinversion could be such that the E7 coding sequences of HPV16 and HPV18are both located between the E6 coding sequences of HPV16 and HPV18. Inparticular the two open reading frames, each with its respectivepromoter, may be arranged next to each other in the recombinant vector.In this case the promoters may be located between the genes, which aretranscribed outwardly, or the promoters may be located outside thegenes, which are transcribed inwardly.

Similarly, the genetic sequences encoding part or all of the E7 proteinfrom HPV16 and the E7 protein from HPV18 may be fused together to form asingle open reading frame. The genetic sequences encoding part or all ofthe E6 protein from HPV16 and the E6 protein from HPV18 may be fusedtogether to form a single open reading frame. This leads to anotherrange of arrangements similar to those shown in FIG. 26. The fusions maybe via a single codon encoding a relatively small neutral amino acide.g. glycine.

Thus the present invention also provides a recombinant virus vectorwhich comprises a first open reading frame having a fuse geneticsequence encoding part or all of the wild-type proteins E6 and E7 fromHPV16; and a separate second open reading frame having a fused geneticsequence encoding part or all of the wild-type proteins E6 and E7 fromHPV18; wherein the first and second open reading frames may be invertedwith respect to one another whereby either: i) the E6 coding sequencesof HPV16 and HPV18 are both located between the E7 coding sequences ofHPV16 and HPV18; or ii) the E7 coding sequences of HPV16 and HPV18 areboth located between the E6 coding sequences of HPV16 and HPV18; andwherein any of said wild-type proteins may be replaced by a mutantprotein immunologically cross-reactive therewith.

Each of the first and second open reading frames may have acorresponding promoter and the two open reading frames each with itspromoter, are arranged next to each other in the virus.

The present invention also provides a recombinant virus vector whereineither: i) the promoters are located between the first and secondreading frames whereby the open reading frames are transcribedoutwardly; or ii) the promoters are located outside the first and secondopen reading frames whereby the open reading frames are transcribedinwardly.

The present invention also provides a recombinant virus vector whichcomprises a first open reading frame having a fused genetic sequenceencoding part or all of the wild-type proteins E6 and E7 from HPV16; anda separate second open reading frame having a fused genetic sequenceencoding part or all of the wild-type proteins E6 and E7 from HPV18;wherein the E6 coding sequences of HPV16 and HPV18 are both locatedbetween the E7 coding sequences of HPV16 and HPV18; and each openreading frame has a corresponding promoter, the promoters being locatedbetween the first and second open reading frames whereby the openreading frames are transcribed outwardly; and wherein any of saidwild-type proteins may be replaced by a mutant protein immunologicallycross-reactive therewith.

The wild-type proteins HPV16E7 and HPV18E7 may be replaced with mutantproteins which are substantially homologous to said wild-type proteinsand in which the residues cys 24 and glu 26 of wild-type protein HPV16E7and the residues cys 27 and glu 29 of wild-type protein HPV18E7 arereplaced with glycine residues.

The recombinant virus vector may be derivable from vaccinia virus.

The applicants have also recognized that for effective function as animmunotherapeutic, it is desirable for the recombinant virus to retainits ability to replicate and thereby generate an active infection inorder that a cellular immune response may be mounted against thevirus-encoded proteins. Thus, the applicants propose that the foreigngene sequences should be inserted into the vector virus at sites, thedisruption of which by the insertion of the heterologous gene sequenceswill not substantially interfere with, and therefore have asubstantially adverse affect on any viral functions which relate to thereplicative ability of the virus in the infected host animal. Theapplicants have named these sites `neutral sites` (although the term`neutral` should not be interpreted strictly as it is acknowledged thatthe disruption of these sites may have a small, but relatively speakinginconsequential adverse affect on replicative ability).

DNA sequences which affect virus replication can fall into severalcategories:

i) protein coding sequences;

ii) elements involved in control of gene expression;

and

iii) elements involved in virus DNA replication A non-essential andneutral insertion site must therefore avoid such regions, and, suchsites have been identified on the basis of nucleotide sequencingstudies. Thus the genetic sequences may be inserted into neutral siteswithin the virus genome. One or more genetic sequences may be insertedinto the same neutral site.

Neutral sites can be easily tested for according to techniques wellknown in the art. For example, a site may be selected, interrupted ordeleted using standard methodologies and the resultant recombinant virusplaced in conditions which normally support growth of the wild typevirus vector, to assess the effect of the manipulations. Thepathogenicity of the virus may be further compared with that of theunmodified virus vector strain in animal models, in order to assess itslevel of attenuation.

In the present invention, the virus vector may be vaccinia virus. Thevaccinia virus may be attenuated or disabled so that it is unable tofully replicate and establish an extensive infection of host cells.

Vaccinia virus has been used extensively in the past for vaccinationagainst smallpox, and its use worldwide has led to the completeeradication of the disease (Bhebehami, Microbiol. Rev., 47, 455, 1983).During the World Health Organisation (WHO) campaign to eradicatesmallpox, several different strains of vaccinia virus were used asvaccines. In 1984 a meeting was sponsored by the WHO to discuss the useof vaccinia virus as live virus vectors (Bulletin of the WHO 63(3):471-477). The data in this report indicates that the number ofcomplications associated with vaccination was lowest for the Wyethstrain of vaccinia virus, and so this strain has been chosen as a basisfor the construction of the recombinant virus according to an embodimentof the present invention.

It is well known that insertion of foreign DNA into the genome ofvaccinia virus at certain favoured sites, such as the thymidine kinasegene locus, can reduce dramatically the ability of the virus toreplicate in vivo. As discussed above, the aim of the therapeuticapproach described here is to generate an active in vivo infection, sothat a cellular immune response may be mounted against the virus encodedproteins. The present invention provides a method for inserting foreigngenes at neutral sites within the genome of a virus, the disruption ofwhich sites by the insertion will not interfere with and therefore havea substantially adverse affect on virus replication.

Where the virus is vaccinia virus, the neutral site may be identifiedherein within the Wyeth strain of vaccinia virus on the basis of therelated WR strain nucleotide sequence. Alternatively, where othervaccinia virus strains are used, sites equivalent to those sitesidentified above may be used. The neutral sites may be any as identifiedhereinafter as A (SEQ ID NO:03), B (SEQ ID NO:04), C (SEQ ID NOS:05),and D (SEQ ID NO:06), or a functional equivalent.

For successful expression of foreign proteins by the recombinant virusvector the foreign genes must be placed under the control of a promotersequence which is operable by the virus. Thus the recombinant virusvector may comprise a single promoter which controls the expression ofall the heterologous genetic sequences within a single open readingframe. Alternatively, where the recombinant virus vector encodes morethan one open reading frame containing heterologous genetic sequences,the virus may comprise a first promoter which controls the expression ofthe genetic sequences from a first open reading frame, and one or morefurther promoters which control the expression of the genetic sequencesfrom one or more further open reading frames. The promoter sequence maybe virus--specific and several have been characterised so far (Davison &Moss, J. Mol. Biol., 210, 749, 1989; Davison & Moss, J. Mol. Biol., 210,771, 1989). The single promoter and the first and one or more furtherpromoters may be the p 7.5 promoter. There have been reports that theinduction of foreign antigen-specific CTLs requires expression of theantigen early in the virus replication cycle (Coupar et al., Eur. J.Immunol., 16, 1479, 1986). Therefore, a recombinant virus as provided bythe present invention may involve the use of the p7.5 promoter(Venkatesan et al., Cell, 125, 805, 1981) and/or the H6 promoter (Roselet al., J. Virol, 60, 436, 1988), both of which are active both earlyand late in infection.

As mentioned earlier, it has been reported that the E7 gene on its ownhas the potential to immortalise cells (Phelps et al., Cell 53, 539,1988). In an embodiment of the present invention, the strategy forexpression of the protein involves production of E7 as a fusion proteinwith E6, which is unlikely to retain biological function. Embodiments ofthe invention provide for reducing this risk still further, by makingchanges within the E7 gene which are known to destroy its oncogeniccapacity (Chesters et al., J. Gen Virol. 71, 449. 1990). Thus in therecombinant virus vectors of the present invention, the geneticsequences encoding part or all of the E7 proteins may be altered fromthe equivalent wild type sequences, in order to render the sequences,used in the recombinant virus vectors less oncogenic than theirequivalent wild type sequences.

The present invention also provides pharmaceuticals comprisingrecombinant virus vectors as herein defined. The pharmaceutical may befor use against a condition caused by HPV infection which comprises animmunotherapeutically effective amount of a recombinant virus vector.The pharmaceutical may be for use against cervical cancer.

The pharmaceutical may be a vaccine to immunise against a conditioncaused by HPV infection which comprises an amount of recombinant virusvector as herein provided which when administered to a recipient canspecifically activate cells of the immune system to HPV proteins. Thevaccine may be for immunisation against cervical cancer.

The pharmaceuticals may comprise one or more excipients. The presentinvention also provides methods of using the recombinant virus vectorsas herein defined to make medicaments for use as immunotherapeutics orvaccines against conditions thought to be caused by HPV infection. Forexample for the prophylaxis and treatment of cervical cancer.

The present invention also provides methods of treating mammalianpatients with recombinant virus vectors and pharmaceuticals as hereinprovided.

The present invention also provides a method of determining a neutralsite in a virus vector, the disruption of which by the insertion ofheterologous gene sequences will not interfere with, and therefore, havea substantially adverse affect on viral function which relates to thereplicative ability of the virus. The method for this determinationcomprises: (a) analysing a viral genome to identify open reading frameswhich are likely to encode functional genes, by looking for expectedcodon usage between spaced apart start and stop codons; and (b)selecting sites which are not in such open reading frames, likely toencode functional genes, as identified in (a) This may include selectingsites between open reading frames for sequences of functional genes andselecting sites which are in open reading frames which have somefunctional gene characteristics, such as an expected codon usage, buthave lost other essential characteristics such as a start codon. Themethod may also comprise interrupting or deleting the selected sitesfrom the viral genome and placing the resultant virus in conditionswhich normally support growth of the wild type virus.

The present invention also provides neutral sites identified by use ofthe above methods.

The present invention provides an embodiment which shows a way ofinducing a cellular immune response against the papillomavirus proteinsusually expressed in cervical tumour cells by the creation of arecombinant vaccinia virus, which has been engineered to produce the HPVE6 and E7 proteins, or proteins containing HPV E6 and E7 sequences,during its replication cycle. This therapeutic vaccinia virus containsthe E6 and E7 genes from both HPV16 and HPV18, the viruses most commonlyassociated with cervical carcinoma. Vaccination with this single virusmay thus stimulate immunity to the E6 and E7 proteins of the HPV typesassociated with more than 80% of cervical tumours. Expression of allfour gene sequences (e.g. HPV16 E6 and E7; HPV18 E6 and E7) in a singlevirus however presents a problem, because of the likelihood of loss ofgenetic sequences through recombination. The present invention providesa method for circumventing this difficulty, firstly through specificsequence alteration, in order to reduce sequence homology and secondlythrough their insertion into the vaccinia virus genome in such a waythat if such recombination were to occur, it would not lead to loss ofsequences (i.e. in inverted orientation with respect to each other).Expression of the desired four gene sequences in the vaccinia virusgenome could also be difficult (though not impossible) to achieve asindependent expression units, and so the invention provides thatinstead, the E6 and E7 open reading frames may be fused together. Aproblem with standard methods for insertion of foreign information intothe vaccinia virus genome is that the use of selectable markers toincrease the efficiency of recombination results in the ultimatepresence in the recombinant virus also of the selectable marker geneitself. Methods for insertion have been developed however, which allowsubsequent elimination of these extraneous sequences (Falkner & Moss J.Virol., 64, 3108, 1990) and these are used in an embodiment of thepresent invention to ensure that the final recombinant vaccinia virushas only those additional sequences which are necessary for its requiredfunction.

All cloning procedures are carried out according to the protocolsdescribed in "Molecular Cloning", A Laboratory Manual, eds. Sambrook,Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989. Allplasmids on which site directed mutagenesis is performed are of the"phagemid" type, which may be converted to single-stranded DNA bysuperinfection with the bacteriophage fl. Preparation and site directedmutagenesis of single-stranded DNA, is carried out as described byBrierley et al., Cell, 57, 537, 1989. The sequence of all the syntheticoligonucleotides used are provided in FIG. 18.

Preparation of the E6 and E7 genes from HPV16 and HPV18 for insertioninto vaccinia virus Cloning of the HPV16 and HPV18 E6 and E7

A fragment of DNA containing the HPV16 E6/7 coding region is prepared bypolymerase chain reaction (PCR) amplification from the plasmidpBR322/HPV16 (Durst et al., PNAS, 80, 3812, 1983) using theoligonucleotides SO5 and SO6. A fragment containing the same region fromHPV18 is prepared by the same procedure from plasmid pBR322/HPV16(Boshart et al., EMBO J. 3,1151) using the oligonucleotides SO1 and S02.Plasmids pBR322/HPV16 and pBR322/HPV18 are both available fromBehringwerke AG, P.O. Box 1140 D-3550, Marburg, Germany (alternativelythe necessary sequences can be created synthetically from the sequenceinformation provided by the present application).

In each case, this produces a DNA fragment of about 800 base pairs (bp)with a site for the restriction enzyme Nco 1 (CCATGG) located exactly atthe beginning of the E6 gene, and a Smal site immediately downstream ofthe termination codon for the E7 gene (FIGS. 1(a) and (b)). The productsare then digested with Ncol and Smal , and cloned into Ncol-Smaldigested plasmid pUC118NS (a modified version of the "phagemid" pUC118(Viera & Messing, Methods Enzymol., 153,3, 1987) in which Ncol and Smalsites have been created by site-directed mutagenesis within thepoly-linker region) to generate the plasmid p1MS7, containing the HPV16sequences, and pIMS8 containing the HPV18 sequences (FIG. 2). The use ofpUC118 is not crucial to the present strategy as any plasmid which canbe manipulated by site directed mutagenesis can be successfully used.

Fusion of the E6 and E7ORFs

For insertion into vaccinia virus, the E6 and E7 genes from each HPVtype, are first fused together to form a single continuous ORF. This isachieved by site-directed mutagenesis as follows:

(i) The termination codon TAA of HPV16 E6 in pIMS7 is altered using theoligonucleotide S20 to the sequence GGAA. This is in order to convertthe normally separate ORFs for HPV16 E6 and E7 into a single ORF(pIMS7.1--FIG. 2).

(ii) The termination codon TAA of HPV18 E6 in pIMS8 is altered using theoligonucleotide S21 to the sequence GGAA. This is in order to convertthe normally separate ORFs for HPV18 E6 and E7 into a single ORF(pIMS8.1--FIG. 2). 5

Abolition of the immortalising potential of E7

In order to destroy the immortalising properties of each of the E7proteins, two key codons within the HPV16 E7 coding sequence, (cys24 andglu26 --FIG. 1(a)) and the equivalent codons from HPV18 E7 (cys27 andglu29--FIG. 1(b)), are altered to glycine residues by site directedmutagenesis as follows.

(i) The sequence of the E7 gene is altered in pIMS7 to encode glycine atcodons 24 and 26 (normally encoding cysteine and glutamate respectively,using oligonucleotide S22 (pIMS7.2--FIG. 2).

(ii) The sequence of the E7 gene is altered in pIMS8 to encode glycineat codons 27 and 29 (normally encoding cysteine and glutamaterespectively, using oligonucleotide S23 (pIMS8.1B --FIG. 2).

Reduction in intertypic recombination potential of HPV16 and HPV18 E6and E7 sequence and elimination of potential vaccinia virustranscription termination signal

A potential difficulty with the presence of both HPV16 and HPV18 E6 andE7 specific DNA within the genome of a single virus, is thatrecombination between the two sets of related sequences could lead toloss or rearrangement of information such that expression of therequired proteins is disrupted. The invention provides ways ofminimising this risk. Firstly, by inserting the two sets of geneticinformation in the vaccinia genome in opposite orientation to each other(so that recombination will result not in the loss of sequenceinformation, but rather in its inversion). Secondly, by creatingspecific changes in the E6/7 sequence of one of the HPV virus strains atsites where the homology is greatest. These changes however are made insuch a way that the amino acid coding potential of the genes remainsunaltered.

The HPV18 E6 sequences is therefore altered by site-directed mutagenesisas follows:

The sequence TTTTTATTCTAGAATTAGAG (SEQ ID NO:01) (which begins 210nucleotides from the start of E6--underlined in FIG. 1(b)) is mutated,using oligonucleotide S24 to the sequence TTTCTACAGTAGAATCAGAG (SEQ IDNO:02)(pIMS8.2--FIG. 2) (changed nucleotides are in bold type).

A second aim of this change is to eliminate from the HPV18 E6 sequence,the sequence TTTTTAT, which is a potential termination signal for theearly vaccinia virus transcription enzyme (Rohrmann et al., Cell., 46,1029, 1986).

Source and propagation of vaccinia virus

The Wyeth strain of vaccinia virus is used for construction of thetherapeutic virus. It is propagated in Vero cells for the purposes ofgenetic manipulation, and in the human diploid fibroblast cell line MRC5for the production of the final therapeutic virus stock. Both cell linesare obtained from the National Institute of Biological Standards andControl, South Mims, U.K. The Wyeth strain of vaccinia virus, Vero cellsand the cell line MRC5 are also available from the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.

Identification and of neutral sites from the vaccinia virus Descriptionof neutral sites

For the purpose of insertion of papillomavirus genes within the vacciniavirus genome, sites have been chosen to have two characteristics.

Firstly, they should be non-essential regions, i.e. insertion of foreigngenes at these points, will not disrupt any functions of the vacciniavirus to the extent that the virus can no longer grow in tissue culture.

Secondly, they should be neutral sites, i.e. insertion of foreign genesall these points, will not increase or decrease the level of attenuationof the vaccinia virus.

The difference between these two factors can be seen by looking at thethymidine kinase (TK) gene of vaccinia virus. It is a non-essentialregion, and hence viruses with genes inserted in the TK gene can growwell in tissue culture (Mackett at al., J. Virol., 49, 857, 1984).However, such viruses have been found to be greatly attenuated in vivo(Buller et al., Nature 317, 813, 1985). For the purpose of prophylacticvaccination, such increased attenuation might be desirable. However, foran immunotherapeutic strategy where the danger from the disease to betreated clearly outweighs the risk of vaccine associated complications,use of an attenuated virus is considered undesirable since it couldcompromise the immunological response to the papillomavirus antigens.Hence, the applicants have identified sites which they judge will notattenuate the virus any further, and have termed them `neutral sites`.Such sites have been identified within the virus genome by carefulanalysis of the DNA sequence of the WR strain. The WR strain wasoriginally derived from the Wyeth strain by passage in mouse brain.Therefore the two strains are closely related. The nucleotide sequenceof three regions of the WR genome which contain the selected neutralsites are shown in FIGS. 19, 20 and 21. Four neutral sites (A-D) havebeen chosen on the above discussed criteria as follows:

Site A: gap between SalF17R and SalF19R

Site B: gap between SalF19R and SalF20.5R

Site C: in SalG2R, a potential non-functional gene Site D: in HindB3.5R,a potential non-functional gene

These sites (A-D) can be identified by the following stretches of DNAsequence, each of which is 40 nucleotides in length.

A CTATCTACCAGATTATTATGTGTTATAAGGTACTTTTTCT (SEQ ID NO:03)

B TATTGTGCTACTGATTCTTCACAGACTGAAGATTGTTGAA (SEQ ID NO:04)

C TCTCTTAAAATGGTTGAGACCAAGCTTCGTTGTAGAAACA (SEQ ID NO:05)

D TGAGGCTACCTCGACATACGTGTGCGCTATCAAAGTGGAA (SEQ ID NO:06)

In other strains these sequences may vary, while still havingsubstantial homology with those given above. In particular a site mayhave at least 90%, more preferably 95%, homology with the sequence givenabove.

FIGS. 3-5 show the distribution of initiation codons and open readingframes (ORFs) in the regions of the vaccinia virus genome shown in FIGS.19, 20 and 21.

FIGS. 6-8 show the same regions with a plot showing to what extent eachreading frame conforms to the pattern of codon usage expected forvaccinia genes. A graph of codon usage is plotted for each of the threepossible reading frames in each direction (Staden, R., Nucl. Acids Res.,12, 521, 1984; Staden, R., Nucl. Acids Res., 12, 551, 1984). In thesecodon usage plots, the short vertical bar lines extending from thehorizontal axes represent start codons. The longer vertical bar linesplaced above the horizontal axes represent stop codons. This sort ofplot is a useful way of helping to determine whether a particular ORF isa genuine vaccinia gene. Where there is a likely genuine gene, the graphof codon usage will rise between a start codon and a stop codon. Forexample, in FIG. 7, it can be seen that the graph of codon usage risesover the region of the SalG2R ORF (the dotted line shows that this frameconforms most of the expected codon usage). For the other two frames thegraphs show that they do not conform to vaccinia codon usage. The peaklabelled `part of gk`, and marked with a dashed line, also conforms wellto vaccinia codon usage. In summary, a genuine gene must start with aninitiation (start) codon, end with a termination (stop) codon, andshould conform well to vaccinia codon usage along its length. In mostcases the conformation to the vaccinia codon usage drops off sharplyoutside the gene. The neutral sites are further described as follows

Site A. Gap between SalF17R and SalF19R

Site A is marked on FIGS. 3 and 6. FIG. 9 (SEQ ID NO:15-17) shows theactual DNA sequence with a translation of the ORFs on either side of thesite. It can be seen that Site A is placed in an intergenic regionbetween SalF17R and SalF19R. It is placed some 195 bases upstream ofSalF19R to avoid any promoter elements associated with that gene. Thesequence TTTTTCT (shown in italics) will act as a terminator of earlyRNA transcription for the SalF17R gene if it is an early gene. However,the site is placed downstream of the first of these, so it will notaffect early termination of transcription if it occurs. Examination ofFIG. 6 shows that there is no recognisable gene on the opposite strandat this point, and hence this sequence location is suitable as a neutralinsertion site.

Site B. Gap between SalF19R and SalF20.5R

Site B is marked on FIGS. 3 and 6. FIG. 10 (SEQ ID NOS:18-20)shows theactual DNA sequence with a translation of the ORFs on either side of thesite. It can be seen that Site B is placed in an intergenic regionbetween SalF19R and SalF20.5R. FIG. 6 shows that it is within a regionof high vaccinia codon usage, but that this region does not form agenuine gene, having no initiation codon. In addition, FIG. 6 suggeststhat Sal20.5R is not a complete gene, as the conformation to vacciniacodon usage drops off dramatically at the start of the gene. In theevent that SalF20.5R is a genuine gene, Site B is placed some 70 basesupstream of SalF20.5R which may well avoid any promoter elementsassociated with that gene. (Note: many vaccinia promoter elements arelocated in approximately 35 bases upstream of the start of the gene.) Inaddition SalF20R has no TTTTTNT (N=any nucleotide) transcriptiontermination signal with which Site B could interfere. Hence thissequence location is suitable as a neutral insertion site.

Site C. Within SalG2R, a potential non-functional gene

Site C is marked on FIG. 4. The ORF SalG2R has considerable similarityto the guanylate kinase (GK) gene of yeast. This similarity is shown inFIG. 11. Sequence upstream of the SalG2R ORF (but in a different frame)has been added on to SalG2R, to see if the match to GK extends beyondthe boundaries of the original open reading frame. The match appears toextend beyond the 5' and of the SalG2R 0RF. In particular, an importantsite in the yeast GK gene, the ATP/GTP binding site (shown underlined)only matches in the out of frame sequence upstream of the SalG2R ORF.Hence, it is very likely that the SalG2R gene is not active as aguanylate kinase and can be referred to as a `pseudogene`. If the geneis inactive as the applicants deduce, then it will serve as a neutralinsertion site.

Site D. Within HindB3.5R, a potential non-functional gene

FIG. 5 shows that site D lies within the region designated HindB3.5R.This region, although conforming to vaccinia codon usage, has no startcodon and is therefore not a genuine gene. The codon usage plot shown inFIG. 8 indicates that it probably was once a functional gene, and maywell have been attached to HindB3R (a shift in the codon usagepreference occurs here well away from the termination codon of theHindB3R ORF which suggests that the last section of HindB3R is notproperly part of this gene.) Hence it is likely that HindB3.5R is notactive as a gene and can be used as a neutral insertion site. FIG. 12(SEQ ID NOS:23-25) shows the actual DNA sequence with a translation ofthe ORFs on either side of the site. It can be seen that site D isplaced in an intergenic region between HindB3R and HindB4R as well asbeing within the non-functional HindB3.5R.

Preparation of vector for cloning of neutral sites

In order to insert foreign genetic information into the neutral sitesdescribed above, DNA copies of the neutral sites, together with anappropriate amount of flanking DNA from the vaccinia genome(approximately 500 bases on either side) must first be cloned into aplasmid vector. These plasmids may then be used to introduce the foreignDNA into the vaccinia virus genome; the vaccinia virus `flankingsequences` around the inserted gene serve to allow homologousrecombination between the plasmid DNA and the viral DNA, with theconsequent insertion of the foreign gene at the desired location.

Cloning of neutral site sequences

Plasmids containing flanking regions from the neutral sites areconstructed as follows. DNA is prepared from the Wyeth strain ofvaccinia virus by the method of Esposito et al., (J. Virol. Meth. 2:175, 1981). The polymerase chain reaction (PCR) is used to remove anapproximately 1000 base pair (bp) fragment from DNA of the Wyeth strainof vaccinia virus. Pairs of oligonucleotides are chosen approximately500 bp either side of the chosen neutral site. These oligonucleotidesare based on the sequence of the WR strain, but are chosen in regionswhere the sequence of the WR strain is identical to that of theCopenhagen strain (Goebel et al., Virology 179:247, 1990). Theoligonucleotides incorporate restriction enzyme recognition sequences sothat they can be cloned easily into a plasmid. For neutral sites A (SEQID NO:03), B (SEQ ID NO:04), and D (SEQ ID NO:06) the restriction sitesare EcoR1 and HindIII. For neutral site C (SEQ ID NO:05) the HindIIIsite is replaced by an Sphl site, since there is an internal HindIIIsite in the chosen flanking sequences.

The oligonucleotides used for PCR are listed below:

Site A (SEQ ID NO:03) leftMB 16

Site A (SEQ ID NO:03) rightMB 17

Site B (SEQ ID NO:04) leftMB 24

Site B (SEQ ID NO:04) rightMB 25

Site C (SEQ ID NO:05) leftMB 18

Site C (SEQ ID NO:05) rightMB 19

Site D (SEQ ID NO:06) leftMB 22

Site D (SEQ ID NO:06) rightMB 23

DNA fragments of approximately lkb are then prepared using these pairsof oligonucleotides by PCR amplification, digested with EcoRI andHindIII (for site A (SEQ NO:03), B (SEQ ID NO:04) and D (SEQ ID NO:06)or with EcoRI and Sphl (for site C (SEQ ID NO:05)) and cloned intoHindIII and EcoRI-digested pUC118 (FIG. 13) to generate the plasmidspIMMC7a, pIMMC7b, pIMMC7c and pIMMC7d.

Creation of unique restriction sites for insertion at the neutral sites

A suitable restriction enzyme site is then introduced at the selectedlocation within each of the plasmids. This is achieved using sitedirected mutagenesis using an oligonucleotide containing the desired newunique site and flanked by 15 bases of sequence to either side (seebelow). The plasmids modified in this fashion are designated pIMMC8a-d(FIG. 13).

    ______________________________________                                        original       oligonucleotidesite new plasmid                                plasmid        introduced                                                     ______________________________________                                        pIMMC7a        MB35SnaB1 pIMMC8a                                              pIMMC7b        MB36Hpa1 pIMMCb                                                pIMMC7c        MB37Stu1 pIMMC8c                                               pIMMC7d        MB38SnaB1 pIMMC8d                                              ______________________________________                                    

Cloning of the vaccinia virus early/late promoter sequences

The p7.5 and H6 promoters from vaccinia virus genomic DNA are preparedby PCR amplification as described below.

A pair of complementary oligonucleotides (S7 and S8) is synthesised toinclude the following restriction enzyme sites, HindIII, Snal Hpal,HindIII, Sall, Ncol, Smal, SnaBl and EcoRl such that the pair, afterannealing, present at one end HindIII compatible overhanging ends, andat the other, EcoRl compatible overhanging ends. The twooligonucleotides are allowed to anneal and are inserted into pUC118 cutwith EcoRl and HindIII (FIG. 14). The resulting vector is called pIMMC3.

A DNA molecule of approximately 180 bp containing the H6 promoter isremoved from the WR strain of vaccinia virus by PCR amplification usingthe oligonucleotides MB15 (anneals upstream and includes a 5'-Sall site)and MB7 (anneals downstream and includes a 5'-HindIII site). This iscloned into pIMMC3 cleaved with HindIII and Sall to create pIMMC4a (FIG.14). A DNA molecule of approximately 200 bp containing the p7.5 promoteris then removed from the WR strain of vaccinia virus by PCRamplification using the oligonucleotides MB32 (anneals upstream andincludes a 5'-Sall site) and MB33 (anneals downstream and includes a5'Ncol site). This is cloned into pIMMC3 cleaved with Ncol and Sall tocreate pIMMC14b.

Construction of the therapeutic virus

The strategy required to generate a recombinant vaccinia viruscontaining and expressing the E6-E7 proteins from HPV16 and HPV18, basedon the elements described above involves five main stages as outlinedbelow.

i) Cloning of the modified E6-7 genes downstream of vaccinia earlypromoter sequences

A DNA fragment containing the modified HPV16 E6-7 sequence is excisedfrom pIMS7.2 by digestion with HindIII and Smal, and cloned into HindIIIand Hpal-digested pIMMC4a to generate pIMS12 (FIG. 15).

A DNA fragment containing the modified HPV18 E6-7 sequence is excisedfrom pIMS8.2 by digestion with Ncol and Smal, and cloned into Ncol andSmal-digested pIMMC14b to generate pIMS14 (FIG. 15).

ii) Preparation of a plasmid vector containing both HPV16 and HPV18 E6-7sequences together with their upstream vaccinia promoters.

A DNA fragment containing the HPV18 E6-7 region together with theupstream p7.5 promoter is excised from pIMS14 with Sall and Smal andinserted into Sall and Smal-digested pIMS12 to generate pIMS15 (FIG. 15)

iii) Insertion of the HPV E6-7/promoter "double" cartridge into theneutral site containing plasmids.

A DNA fragment containing both the HPV16 and HPV18 E6-7 coding regionstogether with their upstream promoter elements is excised from pIMS15with SnaB1 and inserted into the appropriately-digested neutralsite-containing plasmids pIMMC7a-d. This step is shown in FIG. 16, andthe resulting plasmids are designated pIMMC9a-d.

iv) Introduction via homologous recombination of the neutral site DNA,together with the intervening HPV sequences, into the vaccinia virusgenome to create a recombinant virus expressing the two modified HPVE6-7 sequences.

The recombinant plasmids pIMMC9a-d are purified and allowed to recombineinto vaccinia (FIG. 17) using standard protocols (Mackett et al., in D.M. Glover (ed) DNA Cloning: a Practical Approach, Oxford and WashingtonD.C., IRL Press, 1985). Viruses which have acquired the HPV sequencesare identified by probing with radiolabelled HPV specific sequences.Viral plaques are lifted onto nitrocellulose (Villareal and Berg,Science 196, 183, 1977) and probed with radiolabelled NcoI-SmaI fragmentfrom pIMS14 containing the HPV18 E67 gene. Recombinant viruses are thenisolated from the agarose overlay and plaque purified three times. Theyare checked for the presence of the appropriate DNA sequences bySouthern blotting of purified virus DNA using DNA probes derived fromthe HPV E6 and E7 genes, and for expression of the appropriate sequencesby western blotting using antisera specific for the HPV E6 and E7proteins.

Cloning of the therapeutic virus in MRC5 cells

Stocks of the final recombinant virus are prepared by growth in Verocells, and are used to infect MRC5 cells deemed suitable for thepreparation of material suitable for use as human vaccines. The virus isplaque-purified three times by standard methods, and finally a stockprepared for clinical use.

Confirmation of presence of the correct HPV DNA insert

A sample of this stock virus is checked once again for the presence ofcorrectly configured virus DNA, and for expression of the correct virusproteins. FIG. 22 shows the analysis by PCR, of a recombinant vacciniavirus (v9a.1) in which the HPV DNA cassette is inserted at Site A. Thediagram shown in panel (a) indicates the DNA fragments expected ifinsertion of the correct DNA has occurred. It can be seen in panel (b)that the actual pattern of PCR products generated is consistent withthat expected.

Confirmation of expression of the HPV DNA insert

The recombinant viruses are then checked for expression of the expectedHPV proteins. An example of this analysis is shown in FIG. 23. Verocells are infected with recombinant virus v9a.1 (HPV DNA inserted atSite A), and the cells examined by western blotting for the presence ofthe HPV E67 fusions proteins using monoclonal antibodies specific forthe HPV16 E7 protein (camvir3) and for the HPV18 E7 protein (7E10). Itcan be seen that both monoclonal antibodies recognise specificallyproteins of the expected size in cells infected with the recombinantvirus v9a.1, but not in cells infected with the control parent virusWyeth strain. These recognised proteins co-migrate with proteinssynthesised by in vitro translation of mRNA encoding the expected HPVfusion proteins (HPV 16 E67 and HPV18 E67). This experiment indicatessuccessful expression of the heterologous gene sequences from therecombinant virus.

Stability of the HPV DNA insert

For the recombinant virus to be of use clinically, it is important thatthe inserted sequences remain genetically stable over multiple viruspassage, and the DNA insert was carefully designed to promote thisgenetic instability. To confirm the stability of the HPV informationwithin the recombinant virus genome, the virus is subjected to 9 serialpassages, (multiplicity of infection=10 pfu/cell) in Vero cells.Subsequently 20 plaque isolates are picked, and analysed for thepresence of the correct HPV DNA insert by PCR analysis as described inFIG. 22. The data obtained for recombinant virus A are shown in FIG. 24.All 20 virus isolates retained the HPV information in the expectedGenetic arrangement indicating a considerable degree of geneticstability.

Animal experiments

The virulence of the recombinant virus is compared in animal experimentswith that of the parental Wyeth strain. Groups of 20 mice are inoculatedintranasally each with 10⁷ pfu of Wyeth strain or recombinant virus in atotal volume of 20 μl . Two mice are sacrificed at 1 day, 3 days and 5days following inoculation, and the lungs dissected out. The amount ofvirus present in the lungs is then measured by grinding the tissue, andassay of the homogenate by standard vaccinia virus plaque assay. Theresults of such an experiment for the recombinant virus v9a.1 (HPVinfromation inserted at site A (SEQ ID NO:03) are shown in FIG. 25. Itcan be seen that the recombinant virus retains the ability to replicatein mice, and that the level of virus produced in the lungs of theinfected animals is similar to that seen with the parental Wyeth strain.

Therapeutic Use

A stock of the recombinant virus is prepared by infection of MRC5 cells,and adjusted to a concentration of not less than 10⁸ pfu/ml. 20 μl ofthis material is applied to the arm of the patient, which is thenscarified through the virus droplet with a bifurcated needle, accordingto the standard procedure used for vaccination against smallpox.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 70                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTTTTATTCTAGAATTAGAG20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TTTCTACAGTAGAATCAGAG20                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTATCTACCAGATTATTATGTGTTATAAGGTACTTTTTCT40                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TATTGTGCTACTGATTCTTCACAGACTGAAGATTGTTGAA40                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCTCTTAAAATGGTTGAGACCAAGCTTCGTTGTAGAAACA40                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TGAGGCTACCTCGACATACGTGTGCGCTATCAAAGTGGAA40                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 790 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATCCCATGGACCAAAAGAGAACTGCAATGTTTCAGGACCCACAGGAGCGACCCAGAAAGT60                TACCACAGTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGT120               ACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCA180               TAGTATATAGAGATGGGAATCCATATGCTGTATGTGATAAATGTTTAAAGTTTTATTCTA240               AAATTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAAT300               ACAACAAACCGTTGTGTGATTTGTTAATTAGGTGTATTAACTGTCAAAAGCCACTGTGTC360               CTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGT420               GGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCTGT480               AATCATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGAC540               AACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGA600               TGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTGTAACCTTTTGTTG660               CAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTT720               GGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATA780               ACCCGGGTGA790                                                                 (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 263 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..263                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       IleProTrpThrLysArgGluLeuGlnCysPheArgThrHisArgSer                              151015                                                                        AspProGluSerTyrHisSerTyrAlaGlnSerCysLysGlnLeuTyr                              202530                                                                        MetIleXaaTyrXaaAsnValCysThrAlaSerAsnSerTyrCysAsp                              354045                                                                        ValArgTyrMetThrLeuLeuPheGlyIleTyrAlaXaaTyrIleGlu                              505560                                                                        MetGlyIleHisMetLeuTyrValIleAsnValXaaSerPheIleLeu                              65707580                                                                      LysLeuValSerIleAspIleIleValIleValCysMetGluGlnHis                              859095                                                                        XaaAsnSerAsnThrThrAsnArgCysValIleCysXaaLeuGlyVal                              100105110                                                                     LeuThrValLysSerHisCysValLeuLysLysSerLysAspIleTrp                              115120125                                                                     ThrLysSerLysAspSerIleIleXaaGlyValGlyGlyProValAsp                              130135140                                                                     ValCysLeuValAlaAspHisGlnGluHisValGluLysProSerCys                              145150155160                                                                  AsnHisAlaTrpArgTyrThrTyrIleAlaXaaIleTyrValArgPhe                              165170175                                                                     AlaThrArgAspAsnXaaSerLeuLeuLeuXaaAlaIleLysXaaGln                              180185190                                                                     LeuArgGlyGlyGlyXaaAsnArgTrpSerSerTrpThrSerArgThr                              195200205                                                                     GlyGlnSerProLeuGlnTyrCysAsnLeuLeuLeuGlnValXaaLeu                              210215220                                                                     TyrAlaSerValValArgThrLysHisThrArgArgHisSerTyrPhe                              225230235240                                                                  GlyArgProValAsnGlyHisThrArgAsnCysValProHisLeuPhe                              245250255                                                                     SerGluThrIleThrArgVal                                                         260                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 263 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..263                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       SerHisGlyProLysGluAsnCysAsnValSerGlyProThrGlyAla                              151015                                                                        ThrGlnLysValThrThrValMetHisArgAlaAlaAsnAsnTyrThr                              202530                                                                        XaaTyrAsnIleArgMetCysValLeuGlnAlaThrValThrAlaThr                              354045                                                                        XaaGlyIleXaaLeuCysPheSerGlyPheMetHisSerIleXaaArg                              505560                                                                        TrpGluSerIleCysCysMetXaaXaaMetPheLysValLeuPheXaa                              65707580                                                                      AsnXaaXaaValXaaThrLeuLeuLeuXaaPheValTrpAsnAsnIle                              859095                                                                        ArgThrAlaIleGlnGlnThrValValXaaPheValAsnXaaValTyr                              100105110                                                                     XaaLeuSerLysAlaThrValSerXaaArgLysAlaLysThrSerGly                              115120125                                                                     GlnLysAlaLysIleProXaaTyrLysGlySerValAspArgSerMet                              130135140                                                                     TyrValLeuLeuGlnIleIleLysAsnThrXaaArgAsnProAlaVal                              145150155160                                                                  IleMetHisGlyAspThrProThrLeuHisGluTyrMetLeuAspLeu                              165170175                                                                     GlnProGluThrThrAspLeuTyrCysTyrGluGlnLeuAsnAspSer                              180185190                                                                     SerGluGluGluAspGluIleAspGlyProAlaGlyGlnAlaGluPro                              195200205                                                                     AspArgAlaHisTyrAsnIleValThrPheCysCysLysCysAspSer                              210215220                                                                     ThrLeuArgLeuCysValGlnSerThrHisValAspIleArgThrLeu                              225230235240                                                                  GluAspLeuLeuMetGlyThrLeuGlyIleValCysProIleCysSer                              245250255                                                                     GlnLysProXaaProGlyXaa                                                         260                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..182                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ProMetAspGlnLysArgThrAlaMetPheGlnAspProGlnGluArg                              151015                                                                        ProArgLysLeuProGlnLeuCysThrGluLeuGlnThrThrIleHis                              202530                                                                        AspIleIleLeuGluCysValTyrCysLysGlnGlnLeuLeuArgArg                              354045                                                                        GluValTyrAspPheAlaPheArgAspLeuCysIleIleSerGluTyr                              505560                                                                        ArgHisTyrCysTyrSerLeuTyrGlyThrThrLeuGluGlnGlnTyr                              65707580                                                                      GluGluLysGlnArgHisLeuAspLysLysGlnArgPheHisAsnIle                              859095                                                                        ArgGlyArgTrpSerCysMetGluIleHisLeuHisCysMetAsnIle                              100105110                                                                     CysXaaIleCysAsnGlnArgGlnValGlnLeuAspLysGlnAsnArg                              115120125                                                                     ThrGluProIleThrIleLeuXaaProPheValAlaSerValThrLeu                              130135140                                                                     ArgPheGlyCysAlaTyrLysAlaHisThrXaaThrPheValLeuTrp                              145150155160                                                                  LysThrCysXaaTrpAlaHisXaaGluLeuCysAlaProSerValLeu                              165170175                                                                     ArgAsnHisAsnProGly                                                            180                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 817 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ATCCCATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCA60                CGGAACTGAACACTTCACTGCAAGACATAGAAATAACCTGTGTATATTGCAAGACAGTAT120               TGGAACTTACAGAGGTATTTGAATTTGCATTTAAAGATTTATTTGTGGTGTATAGAGACA180               GTATACCCCATGCTGCATGCCATAAATGTATAGATTTTTATTCTAGAATTAGAGAATTAA240               GACATTATTCAGACTCTGTGTATGGAGACACATTGGAAAAACTAACTAACACTGGGTTAT300               ACAATTTATTAATAAGGTGCCTGCGGTGCCAGAAACCGTTGAATCCAGCAGAAAAACTTA360               GACACCTTAATGAAAAACGACGATTTCACAACATAGCTGGGCACTATAGAGGCCAGTGCC420               ATTCGTGCTGCAACCGAGCACGACAGGAACGACTCCAACGACGCAGAGAAACACAAGTAT480               AATATTAAGTATGCATGGACCTAAGGCAACATTGCAAGACATTGTATTGCATTTAGAGCC540               CCAAAATGAAATTCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACTCAGAGGAAGA600               AAACGATGAAATAGATGGAGTTAATCATCAACATTTACCAGCCCGACGAGCCGAACCACA660               ACGTCACACAATGTTGTGTATGTGTTGTAAGTGTGAAGCCAGAATTGAGCTAGTAGTAGA720               AAGCTCAGCAGACGACCTTCGAGCATTCCAGCAGCTGTTTCTGAACACCCTGTCCTTTGT780               GTGTCCGTGGTGTGCATCCCAGCAGTAACCCGGGTGA817                                      (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 272 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..272                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      IleProTrpArgAlaLeuArgIleGlnHisGlyAspProThrSerTyr                              151015                                                                        LeuIleCysAlaArgAsnXaaThrLeuHisCysLysThrXaaLysXaa                              202530                                                                        ProValTyrIleAlaArgGlnTyrTrpAsnLeuGlnArgTyrLeuAsn                              354045                                                                        LeuHisLeuLysIleTyrLeuTrpCysIleGluThrValTyrProMet                              505560                                                                        LeuHisAlaIleAsnValXaaIlePheIleLeuGluLeuGluAsnXaa                              65707580                                                                      AspIleIleGlnThrLeuCysMetGluThrHisTrpLysAsnXaaLeu                              859095                                                                        ThrLeuGlyTyrThrIleTyrXaaXaaGlyAlaCysGlyAlaArgAsn                              100105110                                                                     ArgXaaIleGlnGlnLysAsnLeuAspThrLeuMetLysAsnAspAsp                              115120125                                                                     PheThrThrXaaLeuGlyThrIleGluAlaSerAlaIleArgAlaAla                              130135140                                                                     ThrGluHisAspArgAsnAspSerAsnAspAlaGluLysHisLysTyr                              145150155160                                                                  AsnIleLysTyrAlaTrpThrXaaGlyAsnIleAlaArgHisCysIle                              165170175                                                                     AlaPheArgAlaProLysXaaAsnSerGlyXaaProSerMetSerArg                              180185190                                                                     AlaIleLysArgLeuArgGlyArgLysArgXaaAsnArgTrpSerXaa                              195200205                                                                     SerSerThrPheThrSerProThrSerArgThrThrThrSerHisAsn                              210215220                                                                     ValValTyrValLeuXaaValXaaSerGlnAsnXaaAlaSerSerArg                              225230235240                                                                  LysLeuSerArgArgProSerSerIleProAlaAlaValSerGluHis                              245250255                                                                     ProValLeuCysValSerValValCysIleProAlaValThrArgVal                              260265270                                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 272 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..272                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      SerHisGlyAlaLeuXaaGlySerAsnThrAlaThrLeuGlnAlaThr                              151015                                                                        XaaSerValHisGlyThrGluHisPheThrAlaArgHisArgAsnAsn                              202530                                                                        LeuCysIleLeuGlnAspSerIleGlyThrTyrArgGlyIleXaaIle                              354045                                                                        CysIleXaaArgPheIleCysGlyValXaaArgGlnTyrThrProCys                              505560                                                                        CysMetProXaaMetTyrArgPheLeuPheXaaAsnXaaArgIleLys                              65707580                                                                      ThrLeuPheArgLeuCysValTrpArgHisIleGlyLysThrAsnXaa                              859095                                                                        HisTrpValIleGlnPheIleAsnLysValProAlaValProGluThr                              100105110                                                                     ValGluSerSerArgLysThrXaaThrProXaaXaaLysThrThrIle                              115120125                                                                     SerGlnHisSerTrpAlaLeuXaaArgProValProPheValLeuGln                              130135140                                                                     ProSerThrThrGlyThrThrProThrThrGlnArgAsnThrSerIle                              145150155160                                                                  IleLeuSerMetHisGlyProLysAlaThrLeuGlnAspIleValLeu                              165170175                                                                     HisLeuGluProGlnAsnGluIleProValAspLeuLeuCysHisGlu                              180185190                                                                     GlnLeuSerAspSerGluGluGluAsnAspGluIleAspGlyValAsn                              195200205                                                                     HisGlnHisLeuProAlaArgArgAlaGluProGlnArgHisThrMet                              210215220                                                                     LeuCysMetCysCysLysCysGluAlaArgIleGluLeuValValGlu                              225230235240                                                                  SerSerAlaAspAspLeuArgAlaPheGlnGlnLeuPheLeuAsnThr                              245250255                                                                     LeuSerPheValCysProTrpCysAlaSerGlnGlnXaaProGlyXaa                              260265270                                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 271 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..271                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ProMetAlaArgPheGluAspProThrArgArgProTyrLysLeuPro                              151015                                                                        AspLeuCysThrGluLeuAsnThrSerLeuGlnAspIleGluIleThr                              202530                                                                        CysValTyrCysLysThrValLeuGluLeuThrGluValPheGluPhe                              354045                                                                        AlaPheLysAspLeuPheValValTyrArgAspSerIleProHisAla                              505560                                                                        AlaCysHisLysCysIleAspPheTyrSerArgIleArgGluLeuArg                              65707580                                                                      HisTyrSerAspSerValTyrGlyAspThrLeuGluLysLeuThrAsn                              859095                                                                        ThrGlyLeuTyrAsnLeuLeuIleArgCysLeuArgCysGlnLysPro                              100105110                                                                     LeuAsnProAlaGluLysLeuArgHisLeuAsnGluLysArgArgPhe                              115120125                                                                     HisAsnIleAlaGlyHisTyrArgGlyGlnCysHisSerCysCysAsn                              130135140                                                                     ArgAlaArgGlnGluArgLeuGlnArgArgArgGluThrGlnValXaa                              145150155160                                                                  TyrXaaValCysMetAspLeuArgGlnHisCysLysThrLeuTyrCys                              165170175                                                                     IleXaaSerProLysMetLysPheArgLeuThrPheTyrValThrSer                              180185190                                                                     AsnXaaAlaThrGlnArgLysLysThrMetLysXaaMetGluLeuIle                              195200205                                                                     IleAsnIleTyrGlnProAspGluProAsnHisAsnValThrGlnCys                              210215220                                                                     CysValCysValValSerValLysProGluLeuSerXaaXaaXaaLys                              225230235240                                                                  AlaGlnGlnThrThrPheGluHisSerSerSerCysPheXaaThrPro                              245250255                                                                     CysProLeuCysValArgGlyValHisProSerSerAsnProGly                                 260265270                                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 540 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GATTGTGTGCATGCGTGGTAGATGTTTGGAGAAATGAGAAACTGTTTTCTAGATGGAAAT60                ATTGTTTACGAGCTATTAAACTGTTTATTAATGATCACATGCTTGATAAGATAAAATCTA120               TACTGCAGAATAGACTAGTATATGTGGAAATGTCATAGAAAGTTAAAAGTTAATGAGAGC180               AAAAATATATAAGGTTGTATTCCATATTTGTTATTTTTTCTGTAATAGTTAGAAAAATAC240               ATTCGATGGTCTATCTACCAGATTATTATGTGTTATAAGGTACTTTTTCTCATAATAAAC300               TAGAGTATGAGTAAGATAGTGTTTTTCAAAACATATAAATCTAAAATTGATGGATGAGAT360               ATACAGCTATTAATTTCGAAAATATATTTTAATCTGATAACTTTAAACATGGATTTTTGA420               TGGTGGTTTAACGTTTTAAAAAAAGATTTTGTTATTGTAGTATATGATAATATTAAAAGA480               TGGATATAAAGAATTTGCTGACTGCATGTACTATTTTTTACATTACTACATTGGCTACGG540               (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein (B) LOCATION: 1..52                                     (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      LeuCysAlaCysValValAspValTrpArgAsnGluLysLeuPheSer                              151015                                                                        ArgTrpLysTyrCysLeuArgAlaIleLysLeuPheIleAsnAspHis                              202530                                                                        MetLeuAspLysIleLysSerIleLeuGlnAsnArgLeuValTyrVal                              354045                                                                        GluMetSerXaa                                                                  50                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      MetIleIleLeuLysAspGlyTyrLysGluPheAlaAspCysMetTyr                              151015                                                                        TyrPheLeuHisTyrTyrIleGlyTyrGly                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 540 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TGCCAAGGTTAGATGTAATGGTAACGATAACACAAAATGTGAACGCTGCCCACCTCATAC60                ATATACCACAATCCCAATTATTCTAATGGATGTCATCAATGTAGAAAATGCCCAACCGGA120               TCATTTGATAAGGTAAAGTGTACCGGAACACAGAACAGTAAATGTTCGTGTCTTCCTGGT180               TGGTATTGTGCTACTGATTCTTCACAGACTGAAGATTGTTGAAATTGTGTACCAAAAAGG240               AGATGTCCATGCGGATACTTTGGTGGAATAGATGAACAAGGAAATCCTATTTGTAAATCG300               TGTTGTGTTGGTGAATATTGCGACTACCTACGTAATTATAGACTTGATCCATTTCCTCCA360               TGCAAACTATCTAAATGTAATTAATTATGATTTTGATGATAATGTTACCATACATTATAT420               CGCTACTTGGTTAGTGTATTATTCAGTATGAAGACCTATTAATAATTACTTATCTTTTGA480               CGATCTTGTTATAATTATAATATAAAAATACTTATGGCATAGTAACTCATAATTGCTGAC540               (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AlaLysValArgCysAsnGlyAsnAspAsnThrLysCysGluArgCys                              151015                                                                        ProProHisThrTyrThrThrIleProIleIleLeuMetAspValIle                              202530                                                                        AsnValGluAsnAlaGlnProAspHisLeuIleArgXaa                                       354045                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..60                                                           (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      MetAsnLysGluIleLeuPheValAsnArgValValLeuValAsnIle                              151015                                                                        AlaThrThrTyrValIleIleAspLeuIleHisPheLeuHisAlaAsn                              202530                                                                        TyrLeuAsnValIleAsnTyrAspPheAspAspAsnValThrIleHis                              354045                                                                        TyrIleAlaThrTrpLeuValTyrTyrSerValXaa                                          505560                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      MetSerGlyIleValLysSerIleIleLeuSerGlyProSerGlyLeu                              151015                                                                        GlyLysThrAlaIleAlaLysArgLeuMetGlyIleTyrLeuAspLeu                              202530                                                                        TrpCysProIleProLeuAspPheLeuValLeuMetGluArgGluGly                              354045                                                                        ValAspTyrHisTyrValAsnArgGluAlaIleTrpLysGlyIleAla                              505560                                                                        AlaGlyAsnPheLeuGluHisThrGluPheLeuGlyAsnIleTyrGly                              65707580                                                                      ThrSerLysThrAlaValAsnThrAlaAlaIleAsnAsnArgIleCys                              859095                                                                        ValMetAspLeuAsnIleAspGlyValArgSerLeuLysAsnThrTyr                              100105110                                                                     LeuMetProTyrSerValTyrIleArgProThrSerLeuLysMetVal                              115120125                                                                     GluThrLysLeuArgCysArgAsnThrGluAlaAsnAspGluIleHis                              130135140                                                                     ArgArgValIleLeuAlaLysThrAspMetAspGluAlaAsnGluAla                              145150155160                                                                  GlyLeuPheAspThrIleIleIleGluAspAspValAsnLeuAlaTyr                              165170175                                                                     SerLysLeuIleGlnIleLeuGlnAspArgIleArgMetTyrPheAsn                              180185190                                                                     ThrAsn                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 186 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      SerArgProIleValIleSerGlyProSerGlyThrGlyLysSerThr                              151015                                                                        LeuLeuLysLysLeuPheAlaGluTyrProAspSerPheGlyPheSer                              202530                                                                        ValSerSerThrThrArgThrProArgAlaGlyGluValAsnGlyLys                              354045                                                                        AspTyrAsnPheValSerValAspGluPheLysSerMetIleLysAsn                              505560                                                                        AsnGluPheIleGluTrpAlaGlnPheSerGlyAsnTyrTyrGlySer                              65707580                                                                      ThrValAlaSerValLysGlnValSerLysSerGlyLysThrCysIle                              859095                                                                        LeuAspIleAspMetGlnGlyValLysSerValLysAlaIleProGlu                              100105110                                                                     LeuAsnAlaArgPheLeuPheIleAlaProProSerValGluAspLeu                              115120125                                                                     LysLysArgLeuGluGlyArgGlyThrGluThrGluGluSerIleAsn                              130135140                                                                     LysArgLeuSerAlaAlaGlnAlaGluLeuAlaTyrAlaGluThrGly                              145150155160                                                                  AlaHisAspLysValIleValAsnAspAspLeuAspLysAlaTyrLys                              165170175                                                                     GluLeuLysAspPheIlePheAlaGluLys                                                180185                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CTAAGAACACGTATACGGCAGCAGCTTCCTTTATACTCTCATCTTTTACCAACACAAAGG60                GTGGATATTTGTTCATTGGAGTTGATAATAATACACACAAAGTAATTGGATTCACGGTGG120               GTCATGACTACCTCAGACTGGTAGAGAATGATATAGAAAAGCATATCAAAAGACTTCGTG180               TTGTGCATTTCTGTGAGAAGAAAGAGGACATCAAGTACACGTGTCGATTCATCAAGGTAT240               ATAAACCTGGGGATGAGGCTACCTCGACATACGTGTGCGCTATCAAAGTGGAAAGATGCT300               GTTGTGCTGTGTTTGCAGATTGGCCAGAATCATGGTATATGGATACTAATGGTATCAAGA360               AGTATTCTCCAGATGAATGGGTGTCACATATAAAATTTTAATTAATGTAATAGAGAACAA420               ATAATAAGGTTGTAATATCATATAGACAATAACTAACAATTAATTAGTAACTGTTATCTC480               TTTTTTAACTAACCAACTAACTATATACCTATTAATACATCGTAATTATAGTTCTTAACA540               TCTATTAATCATTAATTCGCTTCTTTAATTTTTTATAAACTAACATTGTTAATTGAAAAG600               GGATAACATGTTACAGAATATAAATTATATATGGATTTTTTTAAAAAGGAAATACTTGAC660               TGGAGTATATATTTATCTCTTCATTATATAGCACGCGTGTTTTCCAATTTTTCCACATCC720               (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..35                                                           (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      LeuArgThrArgIleArgGlnGlnLeuProLeuTyrSerHisLeuLeu                              151015                                                                        ProThrGlnArgValAspIleCysSerLeuGluLeuIleIleIleHis                              202530                                                                        ThrLysXaa                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      MetAspPhePheLysLysGluIleLeuAspTrpSerIleTyrLeuSer                              151015                                                                        LeuHisTyrIleAlaArgValPheSerAsnPheSerThrSer                                    202530                                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TCAGGATCCCACATGAGCGAAAAATACATCG31                                             (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TCAAAGCTTATTACGATACAAACTTAACGGA31                                             (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TCAGTTAACATAAAAAGAACAACGCCCGGCAG32                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TCAAGGGCCTCTATATAGTAATACCAATACTC32                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      TCAGTCGACTTACAAACAACTAGGAAATTGG31                                             (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TCAGAATTCTATGTACAGAGGTCTATTAGGC31                                             (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TCAAAGCTTGTATGAGGTGGGCAGCGTTCAC31                                             (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      TCAGAATTCTTAATTATATTGTCGGCCGTGG31                                             (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      TCAGCATGCATGATCCGTTAGCTTTGGGCTC31                                             (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      TCAGAATTCGAAGCTCTAGAGTATCTTAGCG31                                             (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      TCAAAGCTTTCCTGTATTATATGGGATGTGG31                                             (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      TCAGAATTCATTGATGGATGAGATATACAGC31                                             (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TCAAAGCTTTCACAAAATCG20                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TCAGAATTCCACGTATACGGCAGCAGCTTCC31                                             (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TCAAAGCTTTGTTCTACGTCCATTTTCAAGC31                                             (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TCAGTCGACATACCAATACTCAAGACTACGA31                                             (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      TCACCATGGATTGCTATTGATTGAGTACTGTTC33                                           (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      AGTACCTTATAATACGTAATAATCTGGTAG30                                              (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AATCTTCAGTCTGTTAACAATCAGTAGCAC30                                              (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TACAACGAAGCTAGGCCTCAACCATTTTAA30                                              (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TTTGATAGCGCATACGTATGTCGAGGTAGC30                                              (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AATAGGTATATAGTTAACTGGTTAGTTAAA30                                              (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      ATCCCATGGCGCGCTTTGAGGATCCAAC28                                                (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      TCACCCGGGTTACTGCTGGGATGCACACCAC31                                             (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      ATCCCATGGACCAAAAGAGAACTGCAATGTTTC33                                           (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      TCACCCGGGTTATGGTTTCTGAGAACAGATG31                                             (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      AGCTCTACGTAGTTAACAAGCTTGTCGACCCATGGCCCGGGTACGTA47                             (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      AATTTACGTACCCGGGCCATGGGTCGACAAGCTTGTTAACTACGTAG47                             (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GAAACCCAGCTGGGAATCATGCATGG26                                                  (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GAAACACAAGTAGGAATATTAAGTATG27                                                 (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GATCTCTACGGTTATGGGCAATTAAATGAC30                                              (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GACCTTCTAGGTCACGGGCAATTAAGCGAC30                                              (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      ATGTATAGATTTCTACAGTAGAATCAGAGAATTAAG36                                        (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1250 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      TATTGTTTACGAGCTATTAAACTGTTTATTAATGATCACATGCTTGATAAGATAAAATCT60                ATACTGCAGAATAGACTAGTATATGTGGAAATGTCATAGAAAGTTAAAAGTTAATGAGAG120               CAAAAATATATAAGGTTGTATTCCATATTTGTTATTTTTTCTGTAATAGTTAGAAAAATA180               CATTCGATGGTCTATCTACCAGATTATTATGTGTTATAAGGTACTTTTTCTCATAATAAA240               CTAGAGTATGAGTAAGATAGTGTTTTTCAAAACATATAAATCTAAAATTGATGGATGAGA300               TATACAGCTATTAATTTCGAAAATATATTTTAATCTGATAACTTTAAACATGGATTTTTG360               ATGGTGGTTTAACGTTTTAAAAAAAGATTTTGTTATTGTAGTATATGATAATATTAAAAG420               ATGGATATAAAGAATTTGCTGACTGCATGTACTATTTTTTACATTACTACATTGGCTACG480               GCAGATATACCTACTCCGCCACCAACGGGTCATGTGACAAGGGAGAATATCTTGATAAGA540               GGCATAATCAATGTTGTAATCGGTGTCCACCTGGAGAATTTGCCAAGGTTAGATGTAATG600               GTAACGATAACACAAAATGTGAACGCTGCCCACCTCATACATATACCACAATCCCAATTA660               TTCTAATGGATGTCATCAATGTAGAAAATGCCCAACCGGATCATTTGATAAGGTAAAGTG720               TACCGGAACACAGAACAGTAAATGTTCGTGTCTTCCTGGTTGGTATTGTGCTACTGATTC780               TTCACAGACTGAAGATTGTTGAAATTGTGTACCAAAAAGGAGATGTCCATGCGGATACTT840               TGGTGGAATAGATGAACAAGGAAATCCTATTTGTAAATCGTGTTGTGTTGGTGAATATTG900               CGACTACCTACGTAATTATAGACTTGATCCATTTCCTCCATGCAAACTATCTAAATGTAA960               TTAATTATGATTTTGATGATAATGTTACCATACATTATATCGCTACTTGGTTAGTGTATT1020              ATTCAGTATGAAGACCTATTAATAATTACTTATCTTTTGACGATCTTGTTATAATTATAA1080              TATAAAAATACTTATGGCATAGTAACTCATAATTGCTGACGCGATAAATTCGTAATAATC1140              TGTTTTGTTCAAATTTTTATAAGGAATCTACAGGCATAAAAATAAAAATATAATTTATAA1200              TATACTCTTACAGCGCGCCATCATGAATAACAGCAGTGAATTGATTGCTG1250                        (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 416 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..416                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      TyrCysLeuArgAlaIleLysLeuPheIleAsnAspHisMetLeuAsp                              151015                                                                        LysIleLysSerIleLeuGlnAsnArgLeuValTyrValGluMetSer                              202530                                                                        XaaLysValLysSerXaaXaaGluGlnLysTyrIleArgLeuTyrSer                              354045                                                                        IlePheValIlePheSerValIleValArgLysIleHisSerMetVal                              505560                                                                        TyrLeuProAspTyrTyrValLeuXaaGlyThrPheSerHisAsnLys                              65707580                                                                      LeuGluTyrGluXaaAspSerValPheGlnAsnIleXaaIleXaaAsn                              859095                                                                        XaaTrpMetArgTyrThrAlaIleAsnPheGluAsnIlePheXaaSer                              100105110                                                                     AspAsnPheLysHisGlyPheLeuMetValValXaaArgPheLysLys                              115120125                                                                     ArgPheCysTyrCysSerIleXaaXaaTyrXaaLysMetAspIleLys                              130135140                                                                     AsnLeuLeuThrAlaCysThrIlePheTyrIleThrThrLeuAlaThr                              145150155160                                                                  AlaAspIleProThrProProProThrGlyHisValThrArgGluAsn                              165170175                                                                     IleLeuIleArgGlyIleIleAsnValValIleGlyValHisLeuGlu                              180185190                                                                     AsnLeuProArgLeuAspValMetValThrIleThrGlnAsnValAsn                              195200205                                                                     AlaAlaHisLeuIleHisIleProGlnSerGlnLeuPheXaaTrpMet                              210215220                                                                     SerSerMetXaaLysMetProAsnArgIleIleXaaXaaGlyLysVal                              225230235240                                                                  TyrArgAsnThrGluGlnXaaMetPheValSerSerTrpLeuValLeu                              245250255                                                                     CysTyrXaaPhePheThrAspXaaArgLeuLeuLysLeuCysThrLys                              260265270                                                                     LysGluMetSerMetArgIleLeuTrpTrpAsnArgXaaThrArgLys                              275280285                                                                     SerTyrLeuXaaIleValLeuCysTrpXaaIleLeuArgLeuProThr                              290295300                                                                     XaaLeuXaaThrXaaSerIleSerSerMetGlnThrIleXaaMetXaa                              305310315320                                                                  LeuIleMetIleLeuMetIleMetLeuProTyrIleIleSerLeuLeu                              325330335                                                                     GlyXaaCysIleIleGlnTyrGluAspLeuLeuIleIleThrTyrLeu                              340345350                                                                     LeuThrIleLeuLeuXaaLeuXaaTyrLysAsnThrTyrGlyIleVal                              355360365                                                                     ThrHisAsnCysXaaArgAspLysPheValIleIleCysPheValGln                              370375380                                                                     IlePheIleArgAsnLeuGlnAlaXaaLysXaaLysTyrAsnLeuXaa                              385390395400                                                                  TyrThrLeuThrAlaArgHisHisGluXaaGlnGlnXaaIleAspCys                              405410415                                                                     (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 416 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..416                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      IleValTyrGluLeuLeuAsnCysLeuLeuMetIleThrCysLeuIle                              151015                                                                        ArgXaaAsnLeuTyrCysArgIleAspXaaTyrMetTrpLysCysHis                              202530                                                                        ArgLysLeuLysValAsnGluSerLysAsnIleXaaGlyCysIlePro                              354045                                                                        TyrLeuLeuPhePheLeuXaaXaaLeuGluLysTyrIleArgTrpSer                              505560                                                                        IleTyrGlnIleIleMetCysTyrLysValLeuPheLeuIleIleAsn                              65707580                                                                      XaaSerMetSerLysIleValPhePheLysThrTyrLysSerLysIle                              859095                                                                        AspGlyXaaAspIleGlnLeuLeuIleSerLysIleTyrPheAsnLeu                              100105110                                                                     IleThrLeuAsnMetAspPheXaaTrpTrpPheAsnValLeuLysLys                              115120125                                                                     AspPheValIleValValTyrAspAsnIleLysArgTrpIleXaaArg                              130135140                                                                     IleCysXaaLeuHisValLeuPhePheThrLeuLeuHisTrpLeuArg                              145150155160                                                                  GlnIleTyrLeuLeuArgHisGlnArgValMetXaaGlnGlyArgIle                              165170175                                                                     SerXaaXaaGluAlaXaaSerMetLeuXaaSerValSerThrTrpArg                              180185190                                                                     IleCysGlnGlyXaaMetXaaTrpXaaArgXaaHisLysMetXaaThr                              195200205                                                                     LeuProThrSerTyrIleTyrHisAsnProAsnTyrSerAsnGlyCys                              210215220                                                                     HisGlnCysArgLysCysProThrGlySerPheAspLysValLysCys                              225230235240                                                                  ThrGlyThrGlnAsnSerLysCysSerCysLeuProGlyTrpTyrCys                              245250255                                                                     AlaThrAspSerSerGlnThrGluAspCysXaaAsnCysValProLys                              260265270                                                                     ArgArgCysProCysGlyTyrPheGlyGlyIleAspGluGlnGlyAsn                              275280285                                                                     ProIleCysLysSerCysCysValGlyGluTyrCysAspTyrLeuArg                              290295300                                                                     AsnTyrArgLeuAspProPheProProCysLysLeuSerLysCysAsn                              305310315320                                                                  XaaLeuXaaPheXaaXaaXaaCysTyrHisThrLeuTyrArgTyrLeu                              325330335                                                                     ValSerValLeuPheSerMetLysThrTyrXaaXaaLeuLeuIlePhe                              340345350                                                                     XaaArgSerCysTyrAsnTyrAsnIleLysIleLeuMetAlaXaaXaa                              355360365                                                                     LeuIleIleAlaAspAlaIleAsnSerXaaXaaSerValLeuPheLys                              370375380                                                                     PheLeuXaaGlyIleTyrArgHisLysAsnLysAsnIleIleTyrAsn                              385390395400                                                                  IleLeuLeuGlnArgAlaIleMetAsnAsnSerSerGluLeuIleAla                              405410415                                                                     (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 416 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..416                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      LeuPheThrSerTyrXaaThrValTyrXaaXaaSerHisAlaXaaXaa                              151015                                                                        AspLysIleTyrThrAlaGluXaaThrSerIleCysGlyAsnValIle                              202530                                                                        GluSerXaaLysLeuMetArgAlaLysIleTyrLysValValPheHis                              354045                                                                        IleCysTyrPhePheCysAsnSerXaaLysAsnThrPheAspGlyLeu                              505560                                                                        SerThrArgLeuLeuCysValIleArgTyrPhePheSerXaaXaaThr                              65707580                                                                      ArgValXaaValArgXaaCysPheSerLysHisIleAsnLeuLysLeu                              859095                                                                        MetAspGluIleTyrSerTyrXaaPheArgLysTyrIleLeuIleXaa                              100105110                                                                     XaaLeuXaaThrTrpIlePheAspGlyGlyLeuThrPheXaaLysLys                              115120125                                                                     IleLeuLeuLeuXaaTyrMetIleIleLeuLysAspGlyTyrLysGlu                              130135140                                                                     PheAlaAspCysMetTyrTyrPheLeuHisTyrTyrIleGlyTyrGly                              145150155160                                                                  ArgTyrThrTyrSerAlaThrAsnGlySerCysAspLysGlyGluTyr                              165170175                                                                     LeuAspLysArgHisAsnGlnCysCysAsnArgCysProProGlyGlu                              180185190                                                                     PheAlaLysValArgCysAsnGlyAsnAspAsnThrLysCysGluArg                              195200205                                                                     CysProProHisThrTyrThrThrIleProIleIleLeuMetAspVal                              210215220                                                                     IleAsnValGluAsnAlaGlnProAspHisLeuIleArgXaaSerVal                              225230235240                                                                  ProGluHisArgThrValAsnValArgValPheLeuValGlyIleVal                              245250255                                                                     LeuLeuIleLeuHisArgLeuLysIleValGluIleValTyrGlnLys                              260265270                                                                     GlyAspValHisAlaAspThrLeuValGluXaaMetAsnLysGluIle                              275280285                                                                     LeuPheValAsnArgValValLeuValAsnIleAlaThrThrTyrVal                              290295300                                                                     IleIleAspLeuIleHisPheLeuHisAlaAsnTyrLeuAsnValIle                              305310315320                                                                  AsnTyrAspPheAspAspAsnValThrIleHisTyrIleAlaThrTrp                              325330335                                                                     LeuValTyrTyrSerValXaaArgProIleAsnAsnTyrLeuSerPhe                              340345350                                                                     AspAspLeuValIleIleIleIleXaaLysTyrLeuTrpHisSerAsn                              355360365                                                                     SerXaaLeuLeuThrArgXaaIleArgAsnAsnLeuPheCysSerAsn                              370375380                                                                     PheTyrLysGluSerThrGlyIleLysIleLysIleXaaPheIleIle                              385390395400                                                                  TyrSerTyrSerAlaProSerXaaIleThrAlaValAsnXaaLeuLeu                              405410415                                                                     (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1000 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      ATATTTGGTATTACCGCATTAATTATATTGTCGGCCGTGGCAATTTTCTGTATTACATAT60                TATATATATAATAAACGTTCACGTAAATACAAAACAGAGAACAAAGTCTAGATTTTTGAC120               TTACATAAATGTCTGGGATAGTAAAATCTATCATATTGAGCGGACCATCTGGTTTAGGAA180               AGACAGCCATAGCCAAAAGACTATGGGAATATATTTGGATTTGTGGTGTCCCATACCACT240               AGATTTCCTCGTCCTATGGAACGAGAAGGTGTTGATTACCATTACGTTAACAGAGAGGCC300               ATCTGGAAGGGAATAGCCGCCGGAAACTTTCTAGAACATACTGAGTTTTTAGGAAATATT360               TACGGAACTTCTAAAACAGCTGTGAATACAGCGGCTATTAATAATCGTATTTGTGTGATG420               GATCTAAACATCGACGGTGTTAGAAGTCTTAAAAATACGTACCTAATGCCTTACTCGGTG480               TATATAAGACCTACCTCTCTTAAAATGGTTGAGACCAAGCTTCGTTGTAGAAACACTGAA540               GCTAACGATGAGATTCATCGTCGCGTGATATTGGCAAAAACGGATATGGATGAGGCCAAC600               GAAGCAGGTCTATTCGACACTATTATCATTGAAGATGATGTGAATTTAGCATATAGTAAG660               TTAATTCAGATACTACAGGACCGTATTAGAATGTATTTTAACACTAATTAGAGACTTAAG720               ACTTAAAACTTGATAATTAATAATATAACTCGTTTTTATATGTGGCTATTTCAACGTCTA780               ATGTATTAGTTAAATATTAAAACTTACCACGTAAAACTTAAAATTTAAAATGATATTTCA840               TTGACAGATAGATCACACATTATGAACTTTCAAGGACTTGTGTTAACTGACAATTGCAAA900               AATCAATGGGTCGTTGGACCATTAATAGGAAAAGGTGGATTCGGTAGTATTTATACTACT960               AATGACAATAATTATGTAGTAAAAATAGAGCCCAAAGCTA1000                                  (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 333 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..333                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      IlePheGlyIleThrAlaLeuIleIleLeuSerAlaValAlaIlePhe                              151015                                                                        CysIleThrTyrTyrIleTyrAsnLysArgSerArgLysTyrLysThr                              202530                                                                        GluAsnLysValXaaIlePheAspLeuHisLysCysLeuGlyXaaXaa                              354045                                                                        AsnLeuSerTyrXaaAlaAspHisLeuValXaaGluArgGlnProXaa                              505560                                                                        ProLysAspTyrGlyAsnIlePheGlyPheValValSerHisThrThr                              65707580                                                                      ArgPheProArgProMetGluArgGluGlyValAspTyrHisTyrVal                              859095                                                                        AsnArgGluAlaIleTrpLysGlyIleAlaAlaGlyAsnPheLeuGlu                              100105110                                                                     HisThrGluPheLeuGlyAsnIleTyrGlyThrSerLysThrAlaVal                              115120125                                                                     AsnThrAlaAlaIleAsnAsnArgIleCysValMetAspLeuAsnIle                              130135140                                                                     AspGlyValArgSerLeuLysAsnThrTyrLeuMetProTyrSerVal                              145150155160                                                                  TyrIleArgProThrSerLeuLysMetValGluThrLysLeuArgCys                              165170175                                                                     ArgAsnThrGluAlaAsnAspGluIleHisArgArgValIleLeuAla                              180185190                                                                     LysThrAspMetAspGluAlaAsnGluAlaGlyLeuPheAspThrIle                              195200205                                                                     IleIleGluAspAspValAsnLeuAlaTyrSerLysLeuIleGlnIle                              210215220                                                                     LeuGlnAspArgIleArgMetTyrPheAsnThrAsnXaaArgLeuLys                              225230235240                                                                  ThrXaaAsnLeuIleIleAsnAsnIleThrArgPheTyrMetTrpLeu                              245250255                                                                     PheGlnArgLeuMetTyrXaaLeuAsnIleLysThrTyrHisValLys                              260265270                                                                     LeuLysIleXaaAsnAspIleSerLeuThrAspArgSerHisIleMet                              275280285                                                                     AsnPheGlnGlyLeuValLeuThrAspAsnCysLysAsnGlnTrpVal                              290295300                                                                     ValGlyProLeuIleGlyLysGlyGlyPheGlySerIleTyrThrThr                              305310315320                                                                  AsnAspAsnAsnTyrValValLysIleGluProLysAla                                       325330                                                                        (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 333 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..333                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      TyrLeuValLeuProHisXaaLeuTyrCysArgProTrpGlnPheSer                              151015                                                                        ValLeuHisIleIleTyrIleIleAsnValHisValAsnThrLysGln                              202530                                                                        ArgThrLysSerArgPheLeuThrTyrIleAsnValTrpAspSerLys                              354045                                                                        IleTyrHisIleGluArgThrIleTrpPheArgLysAspSerHisSer                              505560                                                                        GlnLysThrMetGlyIleTyrLeuAspLeuTrpCysProIleProLeu                              65707580                                                                      AspPheLeuValLeuTrpAsnGluLysValLeuIleThrIleThrLeu                              859095                                                                        ThrGluArgProSerGlyArgGluXaaProProGluThrPheXaaAsn                              100105110                                                                     IleLeuSerPheXaaGluIlePheThrGluLeuLeuLysGlnLeuXaa                              115120125                                                                     IleGlnArgLeuLeuIleIleValPheValXaaTrpIleXaaThrSer                              130135140                                                                     ThrValLeuGluValLeuLysIleArgThrXaaCysLeuThrArgCys                              145150155160                                                                  IleXaaAspLeuProLeuLeuLysTrpLeuArgProSerPheValVal                              165170175                                                                     GluThrLeuLysLeuThrMetArgPheIleValAlaXaaTyrTrpGln                              180185190                                                                     LysArgIleTrpMetArgProThrLysGlnValTyrSerThrLeuLeu                              195200205                                                                     SerLeuLysMetMetXaaIleXaaHisIleValSerXaaPheArgTyr                              210215220                                                                     TyrArgThrValLeuGluCysIleLeuThrLeuIleArgAspLeuArg                              225230235240                                                                  LeuLysThrXaaXaaLeuIleIleXaaLeuValPheIleCysGlyTyr                              245250255                                                                     PheAsnValXaaCysIleSerXaaIleLeuLysLeuThrThrXaaAsn                              260265270                                                                     LeuLysPheLysMetIlePheHisXaaGlnIleAspHisThrLeuXaa                              275280285                                                                     ThrPheLysAspLeuCysXaaLeuThrIleAlaLysIleAsnGlySer                              290295300                                                                     LeuAspHisXaaXaaGluLysValAspSerValValPheIleLeuLeu                              305310315320                                                                  MetThrIleIleMetXaaXaaLysXaaSerProLysLeu                                       325330                                                                        (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 333 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..333                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      IleTrpTyrTyrArgIleAsnTyrIleValGlyArgGlyAsnPheLeu                              151015                                                                        TyrTyrIleLeuTyrIleXaaXaaThrPheThrXaaIleGlnAsnArg                              202530                                                                        GluGlnSerLeuAspPheXaaLeuThrXaaMetSerGlyIleValLys                              354045                                                                        SerIleIleLeuSerGlyProSerGlyLeuGlyLysThrAlaIleAla                              505560                                                                        LysArgLeuTrpGluTyrIleTrpIleCysGlyValProTyrHisXaa                              65707580                                                                      IleSerSerSerTyrGlyThrArgArgCysXaaLeuProLeuArgXaa                              859095                                                                        GlnArgGlyHisLeuGluGlyAsnSerArgArgLysLeuSerArgThr                              100105110                                                                     TyrXaaValPheArgLysTyrLeuArgAsnPheXaaAsnSerCysGlu                              115120125                                                                     TyrSerGlyTyrXaaXaaSerTyrLeuCysAspGlySerLysHisArg                              130135140                                                                     ArgCysXaaLysSerXaaLysTyrValProAsnAlaLeuLeuGlyVal                              145150155160                                                                  TyrLysThrTyrLeuSerXaaAsnGlyXaaAspGlnAlaSerLeuXaa                              165170175                                                                     LysHisXaaSerXaaArgXaaAspSerSerSerArgAspIleGlyLys                              180185190                                                                     AsnGlyTyrGlyXaaGlyGlnArgSerArgSerIleArgHisTyrTyr                              195200205                                                                     HisXaaArgXaaCysGluPheSerIleXaaXaaValAsnSerAspThr                              210215220                                                                     ThrGlyProTyrXaaAsnValPheXaaHisXaaLeuGluThrXaaAsp                              225230235240                                                                  LeuLysLeuAspAsnXaaXaaTyrAsnSerPheLeuTyrValAlaIle                              245250255                                                                     SerThrSerAsnValLeuValLysTyrXaaAsnLeuProArgLysThr                              260265270                                                                     XaaAsnLeuLysXaaTyrPheIleAspArgXaaIleThrHisTyrGlu                              275280285                                                                     LeuSerArgThrCysValAsnXaaGlnLeuGlnLysSerMetGlyArg                              290295300                                                                     TrpThrIleAsnArgLysArgTrpIleArgXaaTyrLeuTyrTyrXaa                              305310315320                                                                  XaaGlnXaaLeuCysSerLysAsnArgAlaGlnSerSer                                       325330                                                                        (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1500 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      ACCATCGAGGTAACCACCTCTCTGGAAGACAGCGTGAATAATGTACTCATGAAACGTTTG60                GAAACTATACGCCATATGTGGTCTGTCGTATATGATCATTTTGATATTGTGAATGGTAAA120               GAATGCTGTTATGTGCATACGCATTTGTCTAATCAAAATCTTATACCGAGTACTGTAAAA180               ACAAATTTGTACATGAAGACTATGGGATCATGCATTCAAATGGATTCCATGGAAGCTCTA240               GAGTATCTTAGCGAACTGAAGGAATCAGGTGGATGGAGTCCCAGACCAGAAATGCAGGAA300               TTTGAATATCCAGATGGAGTGGAAGACACTGAATCAATTGAGAGATTGGTAGAGGAGTTC360               TTCAATAGATCAGAACTTCAGGCTGGTGAATCAGTCAAATTTGGTAATTCTATTAATGTT420               AAACATACATCTGTTTCAGCTAAGCAACTAAGAACACGTATACGGCAGCAGCTTCCTTTA480               TACTCTCATCTTTTACCAACACAAAGGGTGGATATTTGTTCATTGGAGTTGATAATAATA540               CACACAAAGTAATTGGATTCACGGTGGGTCATGACTACCTCAGACTGGTAGAGAATGATA600               TAGAAAAGCATATCAAAAGACTTCGTGTTGTGCATTTCTGTGAGAAGAAAGAGGACATCA660               AGTACACGTGTCGATTCATCAAGGTATATAAACCTGGGGATGAGGCTACCTCGACATACG720               TGTGCGCTATCAAAGTGGAAAGATGCTGTTGTGCTGTGTTTGCAGATTGGCCAGAATCAT780               GGTATATGGATACTAATGGTATCAAGAAGTATTCTCCAGATGAATGGGTGTCACATATAA840               AATTTTAATTAATGTAATAGAGAACAAATAATAAGGTTGTAATATCATATAGACAATAAC900               TAACAATTAATTAGTAACTGTTATCTCTTTTTTAACTAACCAACTAACTATATACCTATT960               AATACATCGTAATTATAGTTCTTAACATCTATTAATCATTAATTCGCTTCTTTAATTTTT1020              TATAAACTAACATTGTTAATTGAAAAGGGATAACATGTTACAGAATATAAATTATATATG1080              GATTTTTTTAAAAAGGAAATACTTGACTGGAGTATATATTTATCTCTTCATTATATAGCA1140              CGCGTGTTTTCCAATTTTTCCACATCCCATATAATACAGGATTATAATCTCGTTCGAACA1200              TACGAGAAAGTGGATAAAACAATAGTTGATTTTTTATCTAGGTTGCCAAATTTATTCCAT1260              ATTTTAGAATATGGGGAAAATATTCTACATATTTATTCTATGGATGATGCTAATACGAAT1320              ATTATAATTTTTTTTCTAGATAGAGTATTAAATATTAATAAGAACGGGTCATTTATACAC1380              AATCTCAGGTTATCATCATCCATTAATATAAAAGAATATGTATATCAATTAGTTAATAAT1440              GATCATCCAGATAATAGGATAAGACTAATGCTTGAAAATGGACGTAGAACAAGACATTTT1500              (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 500 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..500                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      ThrIleGluValThrThrSerLeuGluAspSerValAsnAsnValLeu                              151015                                                                        MetLysArgLeuGluThrIleArgHisMetTrpSerValValTyrAsp                              202530                                                                        HisPheAspIleValAsnGlyLysGluCysCysTyrValHisThrHis                              354045                                                                        LeuSerAsnGlnAsnLeuIleProSerThrValLysThrAsnLeuTyr                              505560                                                                        MetLysThrMetGlySerCysIleGlnMetAspSerMetGluAlaLeu                              65707580                                                                      GluTyrLeuSerGluLeuLysGluSerGlyGlyTrpSerProArgPro                              859095                                                                        GluMetGlnGluPheGluTyrProAspGlyValGluAspThrGluSer                              100105110                                                                     IleGluArgLeuValGluGluPhePheAsnArgSerGluLeuGlnAla                              115120125                                                                     GlyGluSerValLysPheGlyAsnSerIleAsnValLysHisThrSer                              130135140                                                                     ValSerAlaLysGlnLeuArgThrArgIleArgGlnGlnLeuProLeu                              145150155160                                                                  TyrSerHisLeuLeuProThrGlnArgValAspIleCysSerLeuGlu                              165170175                                                                     LeuIleIleIleHisThrLysXaaLeuAspSerArgTrpValMetThr                              180185190                                                                     ThrSerAspTrpXaaArgMetIleXaaLysSerIleSerLysAspPhe                              195200205                                                                     ValLeuCysIleSerValArgArgLysArgThrSerSerThrArgVal                              210215220                                                                     AspSerSerArgTyrIleAsnLeuGlyMetArgLeuProArgHisThr                              225230235240                                                                  CysAlaLeuSerLysTrpLysAspAlaValValLeuCysLeuGlnIle                              245250255                                                                     GlyGlnAsnHisGlyIleTrpIleLeuMetValSerArgSerIleLeu                              260265270                                                                     GlnMetAsnGlyCysHisIleXaaAsnPheAsnXaaCysAsnArgGlu                              275280285                                                                     GlnIleIleArgLeuXaaTyrHisIleAspAsnAsnXaaGlnLeuIle                              290295300                                                                     SerAsnCysTyrLeuPhePheAsnXaaProThrAsnTyrIleProIle                              305310315320                                                                  AsnThrSerXaaLeuXaaPheLeuThrSerIleAsnHisXaaPheAla                              325330335                                                                     SerLeuIlePheTyrLysLeuThrLeuLeuIleGluLysGlyXaaHis                              340345350                                                                     ValThrGluTyrLysLeuTyrMetAspPhePheLysLysGluIleLeu                              355360365                                                                     AspTrpSerIleTyrLeuSerLeuHisTyrIleAlaArgValPheSer                              370375380                                                                     AsnPheSerThrSerHisIleIleGlnAspTyrAsnLeuValArgThr                              385390395400                                                                  TyrGluLysValAspLysThrIleValAspPheLeuSerArgLeuPro                              405410415                                                                     AsnLeuPheHisIleLeuGluTyrGlyGluAsnIleLeuHisIleTyr                              420425430                                                                     SerMetAspAspAlaAsnThrAsnIleIleIlePhePheLeuAspArg                              435440445                                                                     ValLeuAsnIleAsnLysAsnGlySerPheIleHisAsnLeuArgLeu                              450455460                                                                     SerSerSerIleAsnIleLysGluTyrValTyrGlnLeuValAsnAsn                              465470475480                                                                  AspHisProAspAsnArgIleArgLeuMetLeuGluAsnGlyArgArg                              485490495                                                                     ThrArgHisPhe                                                                  500                                                                           (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 500 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..500                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      ProSerArgXaaProProLeuTrpLysThrAlaXaaIleMetTyrSer                              151015                                                                        XaaAsnValTrpLysLeuTyrAlaIleCysGlyLeuSerTyrMetIle                              202530                                                                        IleLeuIleLeuXaaMetValLysAsnAlaValMetCysIleArgIle                              354045                                                                        CysLeuIleLysIleLeuTyrArgValLeuXaaLysGlnIleCysThr                              505560                                                                        XaaArgLeuTrpAspHisAlaPheLysTrpIleProTrpLysLeuXaa                              65707580                                                                      SerIleLeuAlaAsnXaaArgAsnGlnValAspGlyValProAspGln                              859095                                                                        LysCysArgAsnLeuAsnIleGlnMetGluTrpLysThrLeuAsnGln                              100105110                                                                     LeuArgAspTrpXaaArgSerSerSerIleAspGlnAsnPheArgLeu                              115120125                                                                     ValAsnGlnSerAsnLeuValIleLeuLeuMetLeuAsnIleHisLeu                              130135140                                                                     PheGlnLeuSerAsnXaaGluHisValTyrGlySerSerPheLeuTyr                              145150155160                                                                  ThrLeuIlePheTyrGlnHisLysGlyTrpIlePheValHisTrpSer                              165170175                                                                     XaaXaaXaaTyrThrGlnSerAsnTrpIleHisGlyGlySerXaaLeu                              180185190                                                                     ProGlnThrGlyArgGluXaaTyrArgLysAlaTyrGlnLysThrSer                              195200205                                                                     CysCysAlaPheLeuXaaGluGluArgGlyHisGlnValHisValSer                              210215220                                                                     IleHisGlnGlyIleXaaThrTrpGlyXaaGlyTyrLeuAspIleArg                              225230235240                                                                  ValArgTyrGlnSerGlyLysMetLeuLeuCysCysValCysArgLeu                              245250255                                                                     AlaArgIleMetValTyrGlyTyrXaaTrpTyrGlnGluValPheSer                              260265270                                                                     ArgXaaMetGlyValThrTyrLysIleLeuIleAsnValIleGluAsn                              275280285                                                                     LysXaaXaaGlyCysAsnIleIleXaaThrIleThrAsnAsnXaaLeu                              290295300                                                                     ValThrValIleSerPheLeuThrAsnGlnLeuThrIleTyrLeuLeu                              305310315320                                                                  IleHisArgAsnTyrSerSerXaaHisLeuLeuIleIleAsnSerLeu                              325330335                                                                     LeuXaaPhePheIleAsnXaaHisCysXaaLeuLysArgAspAsnMet                              340345350                                                                     LeuGlnAsnIleAsnTyrIleTrpIlePheLeuLysArgLysTyrLeu                              355360365                                                                     ThrGlyValTyrIleTyrLeuPheIleIleXaaHisAlaCysPhePro                              370375380                                                                     IlePheProHisProIleXaaTyrArgIleIleIleSerPheGluHis                              385390395400                                                                  ThrArgLysTrpIleLysGlnXaaLeuIlePheTyrLeuGlyCysGln                              405410415                                                                     IleTyrSerIlePheXaaAsnMetGlyLysIlePheTyrIlePheIle                              420425430                                                                     LeuTrpMetMetLeuIleArgIleLeuXaaPhePhePheXaaIleGlu                              435440445                                                                     TyrXaaIleLeuIleArgThrGlyHisLeuTyrThrIleSerGlyTyr                              450455460                                                                     HisHisProLeuIleXaaLysAsnMetTyrIleAsnXaaLeuIleMet                              465470475480                                                                  IleIleGlnIleIleGlyXaaAspXaaCysLeuLysMetAspValGlu                              485490495                                                                     GlnAspIleIle                                                                  500                                                                           (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 500 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..500                                                          (D) OTHER INFORMATION: /note= "Xaa refers to stop codon in                    the open reading frame."                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      HisArgGlyAsnHisLeuSerGlyArgGlnArgGluXaaCysThrHis                              151015                                                                        GluThrPheGlyAsnTyrThrProTyrValValCysArgIleXaaSer                              202530                                                                        PheXaaTyrCysGluTrpXaaArgMetLeuLeuCysAlaTyrAlaPhe                              354045                                                                        ValXaaSerLysSerTyrThrGluTyrCysLysAsnLysPheValHis                              505560                                                                        GluAspTyrGlyIleMetHisSerAsnGlyPheHisGlySerSerArg                              65707580                                                                      ValSerXaaArgThrGluGlyIleArgTrpMetGluSerGlnThrArg                              859095                                                                        AsnAlaGlyIleXaaIleSerArgTrpSerGlyArgHisXaaIleAsn                              100105110                                                                     XaaGluIleGlyArgGlyValLeuGlnXaaIleArgThrSerGlyTrp                              115120125                                                                     XaaIleSerGlnIleTrpXaaPheTyrXaaCysXaaThrTyrIleCys                              130135140                                                                     PheSerXaaAlaThrLysAsnThrTyrThrAlaAlaAlaSerPheIle                              145150155160                                                                  LeuSerSerPheThrAsnThrLysGlyGlyTyrLeuPheIleGlyVal                              165170175                                                                     AspAsnAsnThrHisLysValIleGlyPheThrValGlyHisAspTyr                              180185190                                                                     LeuArgLeuValGluAsnAspIleGluLysHisIleLysArgLeuArg                              195200205                                                                     ValValHisPheCysGluLysLysGluAspIleLysTyrThrCysArg                              210215220                                                                     PheIleLysValTyrLysProGlyAspGluAlaThrSerThrTyrVal                              225230235240                                                                  CysAlaIleLysValGluArgCysCysCysAlaValPheAlaAspTrp                              245250255                                                                     ProGluSerTrpTyrMetAspThrAsnGlyIleLysLysTyrSerPro                              260265270                                                                     AspGluTrpValSerHisIleLysPheXaaLeuMetXaaXaaArgThr                              275280285                                                                     AsnAsnLysValValIleSerTyrArgGlnXaaLeuThrIleAsnXaa                              290295300                                                                     XaaLeuLeuSerLeuPheXaaLeuThrAsnXaaLeuTyrThrTyrXaa                              305310315320                                                                  TyrIleValIleIleValLeuAsnIleTyrXaaSerLeuIleArgPhe                              325330335                                                                     PheAsnPheLeuXaaThrAsnIleValAsnXaaLysGlyIleThrCys                              340345350                                                                     TyrArgIleXaaIleIleTyrGlyPhePheXaaLysGlyAsnThrXaa                              355360365                                                                     LeuGluTyrIlePheIleSerSerLeuTyrSerThrArgValPheGln                              370375380                                                                     PhePheHisIleProTyrAsnThrGlyLeuXaaSerArgSerAsnIle                              385390395400                                                                  ArgGluSerGlyXaaAsnAsnSerXaaPhePheIleXaaValAlaLys                              405410415                                                                     PheIleProTyrPheArgIleTrpGlyLysTyrSerThrTyrLeuPhe                              420425430                                                                     TyrGlyXaaCysXaaTyrGluTyrTyrAsnPhePheSerArgXaaSer                              435440445                                                                     IleLysTyrXaaXaaGluArgValIleTyrThrGlnSerGlnValIle                              450455460                                                                     IleIleHisXaaTyrLysArgIleCysIleSerIleSerXaaXaaXaa                              465470475480                                                                  SerSerArgXaaXaaAspLysThrAsnAlaXaaLysTrpThrXaaAsn                              485490495                                                                     LysThrPhePhe                                                                  500                                                                           __________________________________________________________________________

We claim:
 1. The recombinant vaccinia virus vector which comprises atleast one pair of nucleotide sequences, wherein each of said nucleotidesequences comprises a promoter and an open reading frame which isheterologous to said vaccinia virus and which encodes all or anantigenic part of a human papilloma virus (HPV) protein or animmunologically cross-reactive mutant thereof;wherein said nucleotidesequences of said pair possess a degree of sequence homology with eachother and are inverted with respect to each other in said vaccinia virusvector in order to reduce the risk of recombination which wouldotherwise occur if said sequences were not arranged in an invertedorientation.
 2. The recombinant virus vector according to claim 1wherein the pair of nucleotide sequences encode all or an antigenic partof the HPV wild-type proteins HPV16E7 and HPV18E7 or immunologicallycross-reactive mutants thereof.
 3. The recombinant virus vectoraccording to claim 1 wherein the pair of nucleotide sequences encode allor an antigenic part of the HPV wild-type proteins HPV16E6 and HPV18E6or immunologically cross-reactive mutants thereof.
 4. The recombinantvirus vector according to claim 1 wherein the nucleotide sequences areinserted into the virus vector at one or more neutral sites.
 5. Therecombinant virus vector according to claim 2 which comprises a furtherpair of nucleotide sequences which encode all or an antigenic part ofthe HPV wild-type proteins HPV16E6 and HPV18E6 or immunologicallycross-reactive mutants thereof.
 6. The recombinant virus vectoraccording to claim 1 or 5 wherein two or more nucleotide sequences ofdifferent said pairs are fused together to form a single open readingframe.
 7. The recombinant virus vector according to claim 2 wherein thepair of nucleotide sequences encode all or an antignic part ofimmunological cross-reactive mutants of the wild-type proteins HPV16E7and HPV18E7 and wherein the nucleotide sequence encoding amino acidscorresponding with residues cys 24 and glu 26 of wild-type proteinHPV16E7 or to residues cys 27 and glu 29 of wild-type protein HPV18E7 ischanged so as to encode glycine residues.
 8. The recombinant virusvector according to claim 5 in which the pairs of nucleotide sequencesare arranged in the virus vector according to any one of the options asshown in FIG.
 26. 9. The recombinant virus vector according to claim 6wherein the fusions are via a single codon encoding a small neutralamino acid.
 10. The recombinant virus vector according to claim 9wherein the amino acid is glycine.
 11. The recombinant virus vectoraccording to claim 8 which comprisesa first open reading frame having afused genetic sequence encoding part or all of the wild-type proteins E6and E7 from HPV16; and a separate second open reading frame having afused genetic sequence encoding part or all of the wild-type proteins E6and E7 from HPV18; wherein the first and second open reading frames areinvented with respect to one another whereby either:i) the E6 codingsequences of HPV16 and HPV18 are both located between the E7 codingsequences of HPV16 and HPV18; or ii) the E7 coding sequences of HPV16and HPV18 are both located between the E6 coding sequences of HPV16 andHPV18; and wherein any of said wild-type proteins may be replaced by amutant protein immunologically cross-reactive therewith.
 12. Therecombinant virus vector according to claim 8 which comprisesa firstopen reading frame having a fused genetic sequence encoding all or anantigenic part of the wild-type proteins E6 and E7 from HPV16; and aseparate second open reading frame having a fused genetic sequenceencoding all or an antigenic part of the wild-type proteins E6 and E7from HPV18; wherein the E6 coding sequences of HPV16 and HPV18 are bothlocated between the E7 coding sequences of HPV16 and HPV18; and eachopen reading frame has a corresponding promoter, the promoters beinglocated between the first and second open reading frames whereby theopen reading frames are transcribed outwardly; and wherein any of saidwild-type proteins may be replaced by immunologically cross-reactivemutants thereof.
 13. The recombinant virus vector according to claim 8or 12 wherein either or both of the nucleotide sequences in a said pairof nucleotide sequences comprise nucleotide mutations that do not changethe translated amino acid sequences but make the nucleotide sequencesthemselves less homologous than the corresponding wild-type nucleotidesequences.
 14. The recombiant virus vector according to claim 1, 5, 8,or 12 wherein said heterologous nucleotide sequences may comprise partor all of the sequences shown in FIG. 1(a) and 1(b) (SEQ ID NOS:7-14).15. The recombinant virus vector according to claim 11 wherein each ofthe first and second open reading frames has a corresponding promoterand the two open reading frames each with its promoter, are arrangednext to each other in the virus.
 16. The recombinant virus vectoraccording to claim 15 wherein either:i) the promoters are locatedbetween the first and second open reading frames whereby the openreading frames are transcribed outwardly; or ii) the promoters arelocated outside the first and second open reading frames whereby theopen reading frames are transcribed inwardly.
 17. The recombinant virusvector according to claim 13 wherein the alteration in nucleotidesequence does not result in an alteration of the encoded amino acidsequence.
 18. A recombinant vaccinia virus vector which comprises atleast one pair of nucleotide sequences heterologous to said vacciniavirus, wherein each of said nucleotide sequences encodes all or anantigenic part of a human papillomavirus (HPV) protein or animmunologically cross-reactive mutant thereof; wherein said nucleotidesequences of said pair encode corresponding antigens of different HPVstrains; and wherein said nucleotide sequences of said pair are arrangedin said virus vector such that they are inverted with respect to eachother.
 19. The recombinant vaccinia virus vector according to claim 18wherein the pair of nucleotide sequences encode all or an antigenic partof the HPV wild-type proteins HPV16E7 and HPV18E7 or immunologicallycross-reactive mutants thereof.
 20. The recombinant vaccinia virusvector according to claim 18 wherein the pair of nucleotide sequencesencode all or an antigenic part of the HPV wild-type proteins HPV 16E6and HPV 18E6 or immunologically cross-reactive mutants thereof.
 21. Therecombinant vaccinia virus vector according to claim 18 wherein saidnucleotide sequence are inserted into the virus vector at one or moreneutral sites.
 22. The recombinant vaccinia virus vector according toclaim 4 wherein said one or more neutral sites is selected from siteshaving at least 90% sequence homology with:A) the gap between SallF17Rand SallF19R of strain WR having the sequence

    CTATCTACCAGATTATTATGTGTTATAAGGTACTTTTTCT (SEQ ID NO:03);

B) the gap between SallF19R and SallF20.5R of strain WR having thesequenceti TATTGTGCTACTGATTCTTCACAGACTGAAGATTGTTGAA (SEQ ID NO:04); C) aregion in SallG2R of strain WR having the sequence

    TCTCTTAAAATGGTTGAGACCAAGCTTCGTTGTAGAAACA (SEQ ID NO:05);

D) a region in HindB3.5R of strain WR having the sequence

    TGAGGCTACCTCGACATACGTGTGCGCTATCAAAGTGGAA (SEQ ID NO:06).


23. The recombinant vaccinia virus vector according to claim 19 whichcomprises a further pair of nucleotide sequences which encode all or anantigenic part of the HPV wild-type proteins HPV16E6 and HPV18E6 orimmunologically cross-reactive mutants thereof.
 24. The recombinantvaccinia virus vector according to claim 19 wherein the pair ofnucleotide sequences encode all or an antigenic part of immunologicalcross-reactive mutants of the wild-type proteins HPV16E7 and HPV18E7 andwherein the nucleotide sequence encoding amino acids corresponding toresidues cys 24 and glu 26 of wild-type protein HPV16E7 or to residuescys 27 and glu 29 of wild-type protein HPV18E7 is changed so as toencode glycine residues.
 25. The recombinant vaccinia virus vectoraccording to claim 18 or 23 wherein two or more nucleotide sequences ofsaid different pairs are fused together to form a single open readingframe.
 26. The recombinant vaccinia virus vector according to claim 23in which the pairs of nucleotide sequences are arranged in the virusvector according to any one of the options as shown in FIG.
 26. 27. Therecombinant vaccinia virus vector according to claim 25 wherein thefusions are via a single codon encoding a small neutral amino acid. 28.The recombinant vaccinia virus vector according to claim 27 wherein theamino acid is glycine.
 29. The recombinant vaccinia virus vectoraccording to claim 26 which comprises a first open reading frame havinga fused genetic sequence encoding part or all of the wild-type proteinsE6 and E7 from HPV16; anda separate second open reading frame having afused genetic sequence encoding part or all of the wild-type proteins E6and E7 from HPV18; wherein the first and second open reading frames areinverted with respect to one another whereby either:i) the E6 codingsequences of HPV16 and HPV18 are both located between the E7 codingsequences of HPV16 and HPV18; or ii) the E7 coding sequences of HPV16and HPV18 are both located between the E6 coding sequences of HPV16 andHPV18; and wherein any of said wild-type proteins may be replaced by amutant protein immunologically cross-reactive therewith.
 30. Therecombinant vaccinia virus vector according to claim 26 which comprisesa first open reading frame having a fused genetic sequence encoding allor an antigenic part of the wild-type proteins E6 and E7 from HPV16;anda separate second open reading frame having a fused genetic sequenceencoding all or an antigenic part of the wild-type proteins E6 and E7from HPV18; wherein the E6 coding sequences of HPV16 and HPV18 are bothlocated between the E7 coding sequences of HPV16 and HPV18; and eachopen reading frame has a corresponding promoter, the promoters beinglocated between the first and second open reading frames whereby theopen reading frames are transcribed outwardly; and wherein any of saidwild-type proteins may be replaced by immunologically cross-reactivemutants thereof.
 31. The recombinant vaccinia virus vector according toclaim 26 or 30 wherein either or both of the nucleotide sequences in asaid pair of nucleotide sequences comprise nucleotide mutations that donot change the translated amino acid sequences but make the nucleotidesequences themselves less homologous than the corresponding wild-typenucleotide sequences.
 32. The recombinant vaccinia virus vectoraccording to claim 29 wherein each of the first and second open readingframes has a corresponding promoter and the two open reading frames eachwith its promoter, are arranged next to each other in the virus.
 33. Therecombinant virus vector according to claim 32 wherein either:i) thepromoters are located between the first and second open reading frameswhereby the open reading frames are transcribed outwardly; or ii) thepromoters are located outside the first and second open reading frameswhereby the open reading frames are transcribed inwardly.
 34. Therecombinant vaccinia virus vector according to claim 18, 23, 26, or 30,wherein said heterologous nucleotide sequences comprise part or all ofthe sequences shown in FIGS. 1(a) and 1(b) (SEQ ID NOS:7-14).
 35. Therecombinant vaccinia virus vector according to claim 31 wherein thealteration in nucleotide sequence does not result in an alteration ofthe encoded amino acid sequence.
 36. The recombinant virus vectoraccording to claim 21 which is a recombinant vaccinia virus and whereinsaid one or more neutral sites is selected from sites having at least90% sequence homology with:A) the gap between SallF17R and SallF19R ofstrain WR having the sequence

    CTATCTACCAGATTATTATGTGTTATAAGGTACTTTTTCT (SEQ ID NO:03);

B) the gap between SallF19R and SallF20.5R of strain WR having thesequence

    TATTGTGCTACTGATTCTTCACAGACTGAAGATTGTTGAA (SEQ ID NO:04);

C) a region in SallG2R of strain WR having the sequence

    TCTCTTAAAATGGTTGAGACCAAGCTTCGTTGTAGAAACA (SEQ ID NO:05);

D) a region in HindB3.5R of strain WR having the sequence

    TGAGGCTACCTCGACATACGTGTGCGCTATCAAAGTGGAA (SEQ ID NO:06).


37. A recombinant vaccinia virus vector which comprises at least onepair of nucleotide sequences, wherein each of said nucleotide sequencescomprises a promoter and an open reading frame which is heterologous tosaid vaccinia virus and which encodes all or an antigenic part of ahuman papilloma virus (HPV) protein or an immunologically cross-reactivemutant thereof;wherein said nucleotide sequences of said pair havesequence homology of about 57% or greater whereby the degree of homologyis sufficient for recombination between said sequences to occur; andwherein said nucleotide sequences of said pair are arranged in saidvirus vector such that they are inverted with respect to each other,thereby to reduce the risk of said recombination.